WO2017037593A2

WO2017037593A2 - Pharmaceutical composition used for reducing localised fat and use of pharmaceutical composition

Info

Publication number: WO2017037593A2
Application number: PCT/IB2016/055101
Authority: WO
Inventors: 凌玉芳
Original assignee: 康霈生技股份有限公司; 康霈（美国）股份有限公司
Priority date: 2015-08-28
Filing date: 2016-08-26
Publication date: 2017-03-09
Also published as: US20180243211A1; KR20180039167A; KR102240221B1; JP7063804B2; AU2021202349B2; TW201707686A; CA2995158C; US20180250243A1; AU2016314545C1; TWI667044B; MX2021010359A; JP2018531220A; AU2021202349A1; AU2016314546C1; CA2995158A1; WO2017037594A2; JP7054540B2; TW201806584A; CN108697668B; BR112018003877A2

Abstract

Provided is a pharmaceutical composition used for reducing localised fat, said pharmaceutical composition including a pharmaceutically acceptable aqueous solution, a plurality of drug-containing micelles formed by a surfactant, and resveratrol coated within the drug-containing micelles. The pharmaceutical composition used for reducing localised fat reduces fat at the site of administration, and has advantages such as high stability, high fat tissue bioavailability, low side effects, and sustained release.

Description

Medicinal composition for reducing local fat and use thereof

The present invention relates to a pharmaceutical composition for reducing local fat, and more particularly to a pharmaceutical composition comprising a plurality of drug-containing micelles and resveratrol coated in a drug-containing micelle, and The pharmaceutical composition is used to reduce localized fat. Background technique

In recent years, as more and more people change their perceptions of beauty and improve their standards of self-health and body shape, the issue of concern is no longer simply to reduce weight, but to pay more attention to reducing local fat or sculpture curves. Achieve a healthier and more beautiful look. Moreover, the general method of weight loss, whether it is diet or exercise, can not reduce the fat of a specific single part, to reduce the fat of specific parts (such as the waist, abdomen, legs, arms, chin, and face, etc.) The current technology can only be achieved by means of liposuction.

At present, the method of reducing local fat is mainly liposuction. However, the liposuction process causes serious damage to nerves, blood vessels, and other body tissues, and has infection, large amount of bleeding, excessive anesthesia, and prevention. Fat embolism and the risk of death from anesthesia allergy. In addition, after liposuction, it is also prone to severe redness, severe pain, recovery period of up to 3 months to more than 6 months, and unevenness of liposuction. Therefore, statistics show that although most people want to use liposuction to improve local subcutaneous fat accumulation or body curve, the actual number of people who have undergone liposuction surgery is less than 40%, indicating that most consumers who want to improve the body curve or reduce local fat. , will be abandoned by the side effects of liposuction and postoperative pain or risk.

Although some non-surgical topical fat-reducing pharmaceutical compositions or instruments can reduce some of the side effects, most of them are not effective and cause other side effects, such as necrosis of surrounding normal cells, inflammation of surrounding tissues, And cause severe pain, etc., and there are certain restrictions on the implementation site. Therefore, there is still a lack of a local fat-reducing pharmaceutical composition that is effective in reducing local fat, has fewer side effects, is safer, and has a shorter recovery period.

In the case of high demand from both consumers and physicians, the development of a topical lipid-lowering pharmaceutical composition that is sufficient to break through current technological limitations will be an urgent need to be explored and addressed. Summary of the invention

In view of the deficiencies of the prior art, the present invention provides a pharmaceutical composition for reducing localized fat, comprising a plurality of drug-containing micelles formed by a surfactant, and white coated in the drug-containing micelles Resveratrol. The pharmaceutical composition for reducing local fat can reduce fat at the application site, and has the advantages of high stability, high adipose tissue bioavailability, low side effects, and sustained release.

The invention can promote the apoptotic reaction (apoptosis) of the fat cells at the application site, and achieve the purpose of reducing the local fat at the application site. The invention can greatly improve the conventional technology to make the peripheral cells necrosis (necrosis;), and the adverse reaction of inflammation Should be side effects, and the effect of local fat reduction is significantly better than other non-surgical reduction of local fat pharmaceutical composition. The present invention is suitable for application to a site requiring reduction of subcutaneous fat by transdermal absorption, such as direct injection, subcutaneous implantation, implantable infusion, ointment or patch, without any intervention or assistance of surgery or instrumentation. Preferably, the subcutaneous fat layer is applied to the localized part by subcutaneous fat injection. Preferably, injecting dosage pharmaceutical composition of the present invention include, but are not limited to injection, or injection of ¹ J Qi Qi powder ll (powder for injection, or powder for solution for injection). The topical fat referred to in the present invention includes, but is not limited to, the waist, the abdomen, the legs, the arms, the chin, and the face.

In the present invention, resveratrol refers to resveratrol obtained from natural plant extract or commercially available. Preferably, the resveratrol has a purity of from 90% to 100%.

In the present invention, the green tea extract refers to a mixture of green tea components extracted by any solvent and any extraction method, a commercially available green tea extract, and any one containing at least 45% by weight. ;) a mixture of epigallocatechin gallate (EGCG), or a commercially available epigallocatechin gallate.

In the present invention, a micelle refers to a micro-structure formed by a surfactant having a hydrophilic end and a lipophilic end (lipophilic end), and the surfactant is The micro-structure is formed with the hydrophilic end outward and the lipophilic end (lipophilic end) inward. Preferably, the microstructure is a spherical, spheroidal, or other microstructured structure.

In the present invention, the drug-containing microsphere refers to a resveratrol-containing microcapsule; that is, the drug-containing microsphere refers to a microcapsule coated or containing resveratrol.

In the present invention, the second liposoluble drug microsphere refers to a microcell containing a fat-soluble drug other than resveratrol. That is, the second liposoluble drug microsphere refers to a microcapsule coated or containing other fat-soluble drug.

Wherein, the other lipophilic drug means that at least a lipophilic drug than curcumin (Cur _CU min), quercetin (q _Ue r _C etin), puerarin (puerari _n), and the other resveratrol Or a combination thereof; or, other fat-soluble drugs refer to fat-soluble drugs other than resveratrol.

In the present invention, the water-soluble drug refers to green tea extract, epigallocatechin gallate, epicchinchin, epicchin gallate, Epigallocatechin, Gallocatechin gallate ^ Gallocatechin; Catechin gallate; Catechin; Epigallocatechin gallate (EGCG), caffeine (Caffeine), carnitine (Carnitine; also known as carnitine or carnitine), L-carnitine; At least one or a combination of Synephrine; Chlorogenic acid, and other water soluble drugs.

In the present invention, the term "state in which no precipitate is produced" means any precipitate which is not visible to the human eye, that is, without the use of an artificial device.

The invention provides a pharmaceutical composition for reducing local fat, comprising:

a plurality of drug-containing micelles uniformly distributed in the pharmaceutically acceptable aqueous solution;

Resveratrol coated in the drug-containing micelle;

In a preferred embodiment, the drug-containing micelle is a micro-junction formed by a pharmaceutically acceptable surfactant. The surfactant has a hydrophilic-lipophilic balance value (HLB value;) of more than 10.

In a preferred embodiment, the drug-containing micelles have a particle size of 3 250 nm.

In a preferred embodiment, the drug-containing micelles have a particle size of 5 to 50 nm.

In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable aqueous solution, and the drug-containing micelles are uniformly distributed in the pharmaceutically acceptable aqueous solution. The pharmaceutically acceptable aqueous solution is water for injection, water for injection, or physiological saline.

In a preferred embodiment, the surfactant is a nonionic surfactant.

In a preferred embodiment, the nonionic surfactant is polysorbate 80 (T _ween 80), 2-hydroxyethyl 12-hydroxyoctadecanoate (solutol HS 15), polyoxyethylene castor oil (polyoxyethylene castor oil) At least one or a combination of derivatives;), and other nonionic surfactants.

In a preferred embodiment, the polyoxyethylene castor oil derivative is a polyoxyethylene 35 castor oil (Cremophor ELP;), polyoxyethylene 40 hydrogenated castor oil (Cremophor RH 40), and other polyoxyethylene castor oil derived At least one or a combination thereof.

In a preferred embodiment, the weight ratio of resveratrol to the surfactant is from 1:4 to 1:500.

In a preferred embodiment, the weight ratio of resveratrol to the surfactant is from 1:5 to 1:200.

In a preferred embodiment, the weight ratio of resveratrol to the surfactant is from 1:8 to 1:80.

In a preferred embodiment, the pharmaceutical composition is maintained at an accelerated stability test under conditions of a temperature of 25 ° C ± 2 ° C, a relative humidity of RH 60% ± 5%, and direct light exposure. No precipitate is produced for at least 24 hours.

In a preferred embodiment, the pharmaceutical composition is maintained at an accelerated stability test under conditions of a temperature of 25 ° C ± 2 ° C, a relative humidity of RH 60% ± 5%, and direct light exposure. No sediment is produced for at least 6 months.

In a preferred embodiment, the concentration of resveratrol in the pharmaceutical composition is from 0.2 to 166.7 mg/mL.

In a preferred embodiment, the concentration of resveratrol in the pharmaceutical composition is from 2.5 to 60 mg/mL.

In a preferred embodiment, the pharmaceutical composition further comprises a green tea extract, and the green tea extract is dissolved in the pharmaceutically acceptable aqueous solution; wherein the green tea extract comprises:

A first green tea extract component, wherein the first green tea extract component is epigallocatechin gallate (EGCG).

In a preferred embodiment, the concentration of epigallocatechin gallate in the pharmaceutical composition is from 0.25 to 300. In a preferred embodiment, epigallocatechin gallate is in the pharmaceutical composition. The concentration is 1 200. In a preferred embodiment, the table has the content of gallocatechin gallate, based on the total weight of the green tea extract. In a preferred embodiment, the weight ratio of the resveratrol to the green tea extract is from 30:1 to 1:30.

In a preferred embodiment, the weight ratio of the resveratrol to the green tea extract is from 20:1 to 1:20.

In a preferred embodiment, the weight ratio of the resveratrol to the green tea extract is from 10:1 to 1:10.

In a preferred embodiment, the total weight of the resveratrol and the green tea extract is one unit of weight, and the surfactant has a weight of 0.24 70 units by weight; or is the resveratrol The weight ratio of the total weight of the green tea extract to the surfactant is from 4:1 to 1:70.

In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable aqueous solution and a second liposoluble drug microcapsule, the second liposoluble drug cell being uniformly distributed in the pharmaceutically acceptable aqueous solution in.

The second fat-soluble drug microcell is another micro-structure formed by the second surfactant, and another fat-soluble drug (or a second fat-soluble drug) is coated on the second fat-soluble drug microcapsule in.

In a preferred embodiment, the second surfactant has a hydrophilic-lipophilic balance value (HLB value) greater than 10.

In a preferred embodiment, the second surfactant is a nonionic surfactant.

In a preferred embodiment, the nonionic surfactant is polysorbate 80 (T _ween 80),

2-hydroxyethyl 12-hydroxyoctadecanoate (solutol HS 15), polyoxyethylene castor oil derivative

(polyoxyethylene castor oil derivatives;), and at least one of other nonionic surfactants or combinations thereof.

In a preferred embodiment, the fat-soluble drug is quercetin, synephrine, puerarin, curcumin (C _UrCU mi _n ), and resveratrol At least one or a combination of the fat-soluble drugs.

In a preferred embodiment, the weight ratio of the resveratrol to the second fat-soluble drug is 30 1 to 1 20. In a preferred embodiment, the resveratrol and the second fat-soluble drug are The weight ratio is 20 1 -1. In a preferred embodiment, the weight ratio of the resveratrol to the second fat-soluble drug is 15 1 -1. In a preferred embodiment, the pharmaceutical composition further comprises A pharmaceutically acceptable aqueous solution and a water soluble drug. In a preferred embodiment, the water soluble drug is green tea extract, epigallocatechin gallate, epicchin, Epicachin gallate, Epigallocatechin, Gallocatechin gallate, Gallocatechin, catechin gallate (Catechin) Gallate;), catechin (Catechin;), epigallocatechin gallate (EGCG;), caffeine (Caffeine;), carnitine (Carnitine; also known as carnitine or carni Ting), L-carni (L-carni Tine;), Synephrine;, chlorogenic acid At least one or combination of acid and other water soluble drugs.

In a preferred embodiment, the weight ratio of the resveratrol to the water-soluble drug is from 20:1 to 1:30.

In a preferred embodiment, the weight ratio of the resveratrol to the water-soluble drug is from 15:1 to 1:20.

In a preferred embodiment, the weight ratio of the resveratrol to the water-soluble drug is from 10:1 to 1:15.

In a preferred embodiment, the water-soluble drug is epigallocatechin gallate, and the concentration of epigallocatechin gallate in the pharmaceutical composition is from 0.25 to 300 mg/mL.

In a preferred embodiment, the water-soluble drug is epigallocatechin gallate, and the concentration of epigallocatechin gallate in the pharmaceutical composition is from 1 to 200 mg/mL.

In a preferred embodiment, the water soluble drug is a green tea extract, and the weight ratio of the resveratrol to the green tea extract is from 30:1 to 1:30.

In a preferred embodiment, the water soluble drug is a green tea extract, and the weight ratio of the resveratrol to the green tea extract is from 20:1 to 1:20.

In a preferred embodiment, the water soluble drug is a green tea extract, and the weight ratio of the resveratrol to the green tea extract is from 10:1 to 1:10.

In a preferred embodiment, the pharmaceutical composition further comprises a cosolvent to increase the solubility of the drug. In a preferred embodiment, the co-solvent is at least one of polyethylene glycol, propylene glycol, ethanol, and other co-solvents, or a combination thereof.

In a preferred embodiment, the polyethylene glycol is polyethylene glycol 200 (PEG 200), polyethylene glycol 400 (PEG 400), polyethylene glycol 600 (PEG 600), and other polyethylene glycols. At least one of or a combination thereof.

In a preferred embodiment, the pharmaceutical composition further comprises a suspending agent (also known as a suspending agent;) for reducing the rate of sedimentation of the drug or micelle.

In a preferred embodiment, the suspending agent is sodium alginate, glycerol, carboxymethylcellulose sodium, mannitol, and other suspending agents. At least one or a combination thereof.

In a preferred embodiment, the pharmaceutical composition further comprises oil phase excipients for increasing the stability of the pharmaceutical composition and the solubility of the drug.

In a preferred embodiment, the oil phase excipient is at least one or a combination of an unsaturated fatty acid, glycerol, triglyceride, and other oil phase excipients.

In a preferred embodiment, the unsaturated fatty acid is oleic acid, castor oil, sesame oil, cottonseed oil, soybean oil, red At least one or a combination of safflower oil, corn oil, and other unsaturated fatty acids.

In a preferred embodiment, the triglyceride is at least one of a medium chain triglycerides ^ and other triglycerides or a combination thereof.

In a preferred embodiment, the pharmaceutically acceptable aqueous solution comprises a local anesthetic.

In a preferred embodiment, the local anesthetic is amide, para-aminobenzamine, and amino ether. One less or a combination thereof.

In a preferred embodiment, the amide is Dibucaine, Lidocaine, Mepivacaine HC1, Bupivacine HC1 At least one or a combination of pyrrocaine HCl, prilocaine HCl, Digammacaine, and Oxethazaine.

In a preferred embodiment, the p-aminobenzamine lipid is at least one of or a combination of butacaine (Butacaine;), dimethocaine, and tococaine (Tutocaine) .

In a preferred embodiment, the amino ether is at least one of quinicaine (Quinisocaine;) and pramocaine or a combination thereof.

In a preferred embodiment, the pharmaceutically acceptable aqueous solution contains an antioxidant.

In a preferred embodiment, the antioxidant is beta-carotene, lutein, lycopene, bilirubin, vitamin A (vitamin A) ;), vitamin C (vitamin C; also known as ascorbic acid, ie, vitamin E), uric acid, nitric oxide, nitroxide, pyruvate (pyruvate), catalase; superoxide dismutase; glutathione peroxidases; N-acetylcysteine At least one of cysteine;), and naringenin or a combination thereof.

The present invention further provides a subcutaneous fat layer injection or a hypodermic injection for reducing the amount of local subcutaneous fat, comprising - a pharmaceutically acceptable aqueous solution;

Resveratrol coated in the drug-containing micelle;

Wherein the drug-containing microcapsule is a micro-structure formed by a pharmaceutically acceptable surfactant, and the hydrophilic-lipophilic balance value (HLB value) of the surfactant is greater than 10.

In a preferred embodiment, the amount of topical subcutaneous fat is the amount of subcutaneous fat at the site of application.

In a preferred embodiment, the drug-containing micelles have a particle size of from 3 to 250 nm.

In a preferred embodiment, the pharmaceutically acceptable aqueous solution is water for injection, aqueous solution for injection, or physiological saline.

In a preferred embodiment, the surfactant is a nonionic surfactant.

In a preferred embodiment, the nonionic surfactant is polysorbate 80 (Tween 80),

At least one of 2-hydroxyethyl 12-hydroxyoctadecanoate (solutol HS 15), polyoxyethylene castor oil derivatives, and other nonionic surfactants or combinations thereof.

In a preferred embodiment, the polyoxyethylene castor oil derivative is polyoxyethylene 35 castor oil (Cremophor ELP;), polyoxyethylene 40 hydrogenated castor oil (Cr _e m _O ph _O r RH 40), and others At least one of polyoxyethylene castor oil derivatives Or a combination thereof.

In a preferred embodiment, the concentration of resveratrol in the subcutaneous fat layer injection or subcutaneous injection is

0.2 166.7 mg/mL.

In a preferred embodiment, the subcutaneous fat layer injection or subcutaneous injection further comprises a green tea extract, and the green tea extract is dissolved in the pharmaceutically acceptable aqueous solution; wherein the green tea extract comprises:

a first green tea extract component, wherein the first green tea extract component is epigallocatechin gallate

(epigallocatechin gallate, EGCG).

In a preferred embodiment, the concentration of epigallocatechin gallate in the subcutaneous fat layer injection or subcutaneous injection is 0.25 to 300 mg/mL.

In a preferred embodiment, the concentration of epigallocatechin gallate in the subcutaneous fat layer injection or subcutaneous injection is from 1 to 200 mg/mL.

In a preferred embodiment, the epigallocatechin gallate is present in an amount of from 45 to 100% by weight based on 100% by weight of the total of the green tea extract.

In a preferred embodiment, the weight ratio of the resveratrol to the green tea extract is from 30:1 to 1:30.

In a preferred embodiment, the weight ratio of the resveratrol to the green tea extract is from 20:1 to 1200.

In a preferred embodiment, the weight ratio of the resveratrol to the green tea extract is from 10:1 to 1 10 .

In a preferred embodiment, the application site of the application site is: 0.2 per square centimeter injection.

In a preferred embodiment, the application site of the application site: 0.4 injection per square centimeter

In a preferred embodiment, the application site of the application site is 0.240 mg per kg.

In a preferred embodiment, the application site of the application site is 0.4 to 20 mg per kg.

In a preferred embodiment, the application frequency is from 1 to 12 administrations per interval from 1 day to 30 days. In a preferred embodiment, the application frequency is from 1 to 8 times per interval from 1 day to 21 days. In a preferred embodiment, the subcutaneous fat layer injection or subcutaneous injection further comprises a second fat-soluble drug microcapsule, and the second fat-soluble drug microcell is uniformly distributed in the pharmaceutically acceptable aqueous solution. in.

The second fat-soluble drug micelle is a micro-structure formed by another surfactant, and the other fat-soluble drug is coated in the second fat-soluble drug micelle.

In a preferred embodiment, the hydrophilic-lipophilic balance of the other surfactant The balance value, HLB value is greater than 10.

In a preferred embodiment, the additional surfactant is a nonionic surfactant.

Preferably, the fat-soluble drug is at least one or a combination of curcumin, quercetin, puerarin, and other fat-soluble drugs other than resveratrol.

In a preferred embodiment, the pharmaceutically acceptable aqueous solution further comprises a water soluble drug.

In a preferred embodiment, the water-soluble drug is green tea extract, epigallocatechin gallate, epicchinchin, epicatechin gallate, and epicatechin gallate. ;), Epigallocatechin, Gallocatechin gallate ^ Gallocatechin; Catechin gallate; Catechin ;), epigallocatechin gallate (EGCG), caffeine (Caffeine), carnitine (Carnitine; also known as carnitine or carnitine), L-carnitine; At least one or a combination of, Synephrine; Chlorogenic acid, and other water soluble drugs.

In a preferred embodiment, the local anesthetic is at least one of an amide, a p-aminobenzamide, and an amino ether, or a combination thereof.

In a preferred embodiment, the p-aminobenzamide is at least one or a combination of butacaine (Butacaine;), dimethocaine, and Tutocaine.

In a preferred embodiment, the antioxidant is beta-carotene, lutein, lycopene, bilirubin, vitamin A (vitamin A) ;), vitamin C (vitamin C; also known as ascorbic acid, ie, vitamin E), uric acid, nitric oxide, nitroxide, pyruvate (pyruvate), catalase; superoxide dismutase (superoxide) At least one of or a combination of glutathione peroxidases, N-acetyl cysteine, and naringenin.

The present invention provides a method of reducing the amount of subcutaneous fat in a localized portion of an individual comprising administering a pharmaceutical composition to the localized portion of the individual, wherein the pharmaceutical composition comprises:

a plurality of drug-containing micelles;

Resveratrol coated in the drug-containing micelle;

Wherein the drug-containing microcapsule is a picostructure formed by a pharmaceutically acceptable surfactant, and the hydrophilic-lipophilic balance value (HLB value) of the surfactant is greater than 10.

In a preferred embodiment, the surfactant is a nonionic surfactant.

In a preferred embodiment, wherein the weight ratio of resveratrol to the surfactant is from 1:4 to 1:500. In a preferred embodiment, wherein the weight ratio of resveratrol to the surfactant is 1:

In a preferred embodiment, wherein the weight ratio of resveratrol to the surfactant is from 1:8 to 1:80

o In a preferred embodiment, wherein the concentration of resveratrol in the pharmaceutical composition is from 0.2 to 166.7 mg/m oL. In a preferred embodiment, wherein the concentration of resveratrol in the pharmaceutical composition is from 2.5 to 60 mg/mL. In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable aqueous solution and a second liposoluble drug microcapsule, the second liposoluble drug cell is uniformly distributed in the pharmaceutically acceptable In the aqueous solution; wherein the second fat-soluble drug micelle is another micro-shaped structure formed by the second non-ionic surfactant, and a second fat-soluble drug is coated on the second fat-soluble drug In the microcells.

In a preferred embodiment, the second nonionic surfactant has a hydrophilic-lipophilic balance value (HLB value) greater than 10.

In a preferred embodiment, the second nonionic surfactant is polysorbate 80 (T _ween 80),

In a preferred embodiment, the second nonionic surfactant is polyoxyethylene 35 castor oil (Cremophor ELP), polyoxyethylene 40 hydrogenated castor oil (Cremophor RH 40), and other polyoxyethylene castor oil derived To One less or a combination thereof.

In a preferred embodiment, the second fat-soluble drug is quercetin, synephrine, puerarin, curcuminoid, curcumin, and At least one or a combination of other fat-soluble drugs other than resveratrol.

In a preferred embodiment, the weight ratio of the resveratrol to the second fat-soluble drug is: In a preferred embodiment, the weight ratio of the resveratrol to the second fat-soluble drug is: In a preferred embodiment, the weight ratio of the resveratrol to the second fat-soluble drug is: In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable aqueous solution and a water-soluble drug. In a preferred embodiment, the water-soluble drug is a green tea extract, epigallocatechin gallate. Acidatelocatechin gallate, epicchinchin, epicchinchin gallate, Epigallocatechin, Gallocatechin gallate, gallnut Calocatechin;, catechin gallate; Catechin; Cepoch; epigallocatechin gallate (EGCG;), caffeine (Caffeine;) Carnitine (also known as carnitine or carnitine), L-carnitine, Synephrine, Chlorogenic acid, and other water-soluble drugs At least one of or a combination thereof.

In a preferred embodiment, the weight ratio of the resveratrol to the water-soluble drug is 20

In a preferred embodiment, the weight ratio of the resveratrol to the water-soluble drug is 20. In a preferred embodiment, the weight ratio of the resveratrol to the water-soluble drug is

In a preferred embodiment, the water-soluble drug is epigallocatechin gallate, and the concentration of epigallocatechin gallate in the pharmaceutical composition is 0.25 300 mg/mL.

In a preferred embodiment, the water-soluble drug is epigallocatechin gallate, and the concentration of epigallocatechin gallate in the pharmaceutical composition is mg/mL.

In a preferred embodiment, the water soluble drug is a green tea extract, and the weight ratio of the resveratrol to the green tea extract is .

In a preferred embodiment, the water-soluble drug is a green tea extract, and the weight ratio of the resveratrol to the green tea extract is: to : .

In a preferred embodiment, the total weight of the resveratrol and the green tea extract is one unit of weight, and the surfactant has a weight of 0.24 70 units by weight; or is the resveratrol The weight ratio of the total weight of the green tea extract to the surfactant is up to.

In a preferred embodiment, the pharmaceutical composition is administered at a localized dose of 0.2 to 20 mg per square centimeter.

In a preferred embodiment, the dose of the pharmaceutical composition applied at the local site is 0.4 ~ per square centimeter. In a preferred embodiment, the pharmaceutical composition is administered at a localized dose of 0.2 to 40 mg per kilogram. In a preferred embodiment, the pharmaceutical composition is applied at a localized dose of 0.4 to 20 mg per kg. In a preferred embodiment, the frequency of administration of the pharmaceutical composition is from 1 to 12 times per one to one day interval.

In a preferred embodiment, the frequency of administration of the pharmaceutical composition is administered from 1 to 8 times per interval from 1 day to 21 days.

In a preferred embodiment, the individual is an animal or a human.

In a preferred embodiment, the pharmaceutical composition is injected, or applied to the localized portion of the individual. In a preferred embodiment, the pharmaceutical composition further comprises at least one of a cosolvent, a suspending agent, and an oil phase excipients; combination.

In a preferred embodiment, the oil phase excipient and/or co-solvent together with the nonionic surfactant form the microstructure.

The present invention provides a pharmaceutical composition for the preparation of a medicament or a subcutaneous injection formulation for reducing the amount of subcutaneous fat in a localized part of an individual; the pharmaceutical composition comprising:

a plurality of drug-containing micelles;

Resveratrol coated in the drug-containing micelle;

Wherein the drug-containing microsphere is a micro-structure formed by a pharmaceutically acceptable nonionic surfactant, and the hydrophilic-lipophilic balance value (HLB) of the nonionic surfactant Value ;) is greater than ιο.

In a preferred embodiment, the weight ratio of resveratrol to the nonionic surfactant is from 1:4 to 1:500. In a preferred embodiment, the concentration of resveratrol in the pharmaceutical composition is from 0.2 to 166.7 mg/mL.

In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable aqueous solution and a second fat-soluble drug microcell, the second liposoluble drug microcell is uniformly distributed in the pharmaceutically acceptable In the aqueous solution; wherein the second fat-soluble drug micelle is another micro-shaped structure formed by the second non-ionic surfactant, and a second fat-soluble drug is coated on the second fat-soluble drug micro In the cell. In a preferred embodiment, the second nonionic surfactant has a hydrophilic-lipophilic balance value (HLB value) greater than 10.

In a preferred embodiment, the second nonionic surfactant is polysorbate 80 (T _ween 80), 2-hydroxyethyl 12-hydroxyoctadecanoate (solutol HS 15), polyoxyethylene castor oil derivative (polyoxyethylene) At least one of castor oil derivatives;), and other nonionic surfactants, or a combination thereof.

In a preferred embodiment, the second nonionic surfactant is polyoxyethylene 35 castor oil (Cremophor ELP) ^ polyoxyethylene 40 hydrogenated castor oil (Cremophor RH 40), and other polyoxyethylene castor oil derived At least one or a combination thereof.

In a preferred embodiment, the second fat-soluble drug is quercetin, synephrine, puerarin, curcuminoid, and other resveratrol. At least one or a combination of the other fat-soluble drugs.

In a preferred embodiment, the weight ratio of the resveratrol to the second fat-soluble drug is from 30:1 to 1:20. In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable aqueous solution and a water soluble drug.

In a preferred embodiment, the water-soluble drug is green tea extract, Epigallocatechin gallate, Epicatechin, Epicatchin gallate, Epigallocatechin, Gallocatechin gallate, Gallocatechin; Catechin gallate; Catechin; Epigallocatechin gallate (EGCG;), caffeine (Caffeine;), carnitine (Carnitine; also known as carnitine or carnitine), L-carnitine; At least one or a combination of, Synephrine; Chlorogenic acid, and other water soluble drugs.

In a preferred embodiment, the water-soluble drug is epigallocatechin gallate, and the concentration of epigallocatechin gallate in the subcutaneous fat layer injection or subcutaneous injection is 0.25-300 mg/ mL.

In a preferred embodiment, the total weight of the resveratrol and the green tea extract is one unit of weight, and the weight of the nonionic surfactant is 0.24 70 units by weight; or alternatively, the chalk The weight ratio of the total weight of rusol to the green tea extract to the nonionic surfactant is from 4:1 to 1:70.

In a preferred embodiment, the pharmaceutical or subcutaneous injection comprises a therapeutically effective amount of the pharmaceutical composition.

In a preferred embodiment, the therapeutically effective amount is from 0.2 to 20 mg per square centimeter of the topical portion. The pharmaceutical composition is in a preferred embodiment, and a therapeutically effective amount is 0.2 40 mg of the pharmaceutical composition per kg of body weight.

In a preferred embodiment, the drug or subcutaneous injection is administered at a frequency of from 1 day to 30 days per interval. Use the site 1 to 12 times.

In a preferred embodiment, the individual is an animal.

In a preferred embodiment, the drug or subcutaneous injection is administered to the localized portion of the individual; or the drug is applied to the localized portion of the individual.

In a preferred embodiment, the pharmaceutical composition further comprises a cosolvent, a suspending agent, and at least one or a combination of oil phase excipients; .

In a preferred embodiment, the oil phase excipient and/or co-solvent together with the nonionic surfactant form the microstructure. DRAWINGS

Figure 1A: Bar graph of the effect of subcutaneous injection of resveratrol without microvesicles and subcutaneous injection of green tea extract without microvesicles on subcutaneous fat mass in rats.

Figure 1B: Bar graph of the effect of murine-free resveratrol subcutaneous injection and micronucleated green tea extract subcutaneous injection on relative total weight gain in rats.

Figure 2A: Bar graph of the effect of subcutaneous injection of resveratrol prepared by different excipients on local subcutaneous fat mass in rats.

Figure 2B: Bar graph of the effect of subcutaneous injection of resveratrol prepared by different excipients on relative total weight gain in rats.

Figure 3: Bar graph of the effect of resveratrol pharmaceutical composition on subcutaneous fat mass in rats.

Figure 4A: Bar graph of the effect of resveratrol combination medicinal composition on subcutaneous fat mass in rats.

Figure 4B: Bar graph of the effect of resveratrol combination medicinal composition on relative total weight gain in rats.

Figure 5: Effect of resveratrol-other fat-soluble drug combination medicinal composition on apoptosis of mature adipocytes.

Figure 6: Effect of resveratrol - other water-soluble drug combination medicinal composition on apoptosis of mature adipocytes. detailed description

Resveratrol is a polyphenolic compound found mainly in red grape skins, knotweed, or red wine. Resveratrol is used because resveratrol is difficult to dissolve in aqueous solution, is easily metabolized in the body to glucuronic acid and sulfate metabolites, is rapidly excreted in urine and feces, and its body availability is extremely poor. The development of pharmaceutical compositions that reduce localized fats faces considerable difficulties. The inventor faced this dilemma at the beginning of the development of this pharmaceutical composition.

Experiment 1: Effect of subcutaneous injection of resveratrol and green tea extract on subcutaneous fat and body weight of rats. Preparation of green tea extract subcutaneous injection: Green tea extract was prepared into 5 mg/mL using water for injection. Aqueous solution. It was filtered through a 0.2 μηη filter, which was the subcutaneous injection of 5 mg/mL green tea extract described in this experiment. Store in a refrigerator at 4 °C in the dark. Preparation of resveratrol subcutaneous injection: Resveratrol is formulated into 5 by using polysorbate 80 (Tween 80), solvent ethanol, and water for injection, which are commonly used for water-insoluble pharmaceutical injections. Mg/mL solution. The detailed preparation method is as follows: 0.5 g of resveratrol is mixed with an appropriate amount of solvent ethanol to completely dissolve the resveratrol, and then 0.1 g of polysorbate 80 (Tween 80) is added to completely dissolve the polysorbate; The gas is volatilized for 2 to 4 hours to volatilize the ethanol. After the ethanol is completely evaporated, the water for injection is added to make the total volume 100 mL. After stirring evenly, the membrane is filtered with a 0.2 μηη filter, which is 5 mg/ of the experiment. mL of resveratrol subcutaneous injection. Store in a refrigerator at 4 °C in the dark.

Experiments were performed using a 7-week old SD strain male rat (male Sprague-Dawley rat). First, 18 rats were fed with high-fat diet (Research Diets, Inc.; model #1) 12492;) to induce subcutaneous fat booster B, and continuously fed to rats weighing 330± After 10 g, the rats were randomly divided into three groups: a high-fat control group, a resveratrol group, and a green tea extract group, with 6 rats in each group, so that there was no statistical difference in body weight between the groups. The body weight of each rat was recorded and defined as the "pre-test weight" of each rat. Then, the drug is administered in the following manner.

5 mg/mL of resveratrol subcutaneous injection was injected into the subcutaneous fat layer of the inguinal region of rats in the resveratrol group, and each injection was injected with 8 mg (8 mg/kg) of chalk per kg body weight. Resveratrol; that is, 1.6 mL of the above 5 mg/mL resveratrol subcutaneous injection per kilogram of body weight. 5 mg/mL of green tea extract was injected subcutaneously into the subcutaneous fat layer of the inguinal region of the green tea extract group, and 8 mg (8 mg/kg) of green tea extract per kg body weight was injected per injection; That is, 1.6 mL of the above 5 mg/mL green tea extract subcutaneous injection was injected per kilogram of body weight. In the high-fat control group, the same volume of water for injection was administered in the same manner as described above.

The above injection site was the lower inguinal fat of rats, which was injected on the left and right sides on average, and was injected once on the first, third and fifth days of the test. High-fat diets were continuously administered during the test, and changes in body weight were recorded daily. Drinking water was recorded once a week, fasted on the 20th day of the test, and rats were sacrificed with carbon dioxide on the 21st day.

The body weight of each rat was recorded and defined as the "post-test weight" of each rat. The "post-test weight" was subtracted from the "post-test weight" of each rat to obtain "total weight gain". The total weight gain of each group of rats was divided by the total weight gain of the rats in the high-fat control group to obtain "relative total weight gain".

The fat in the lower groin of the left and right sides of the rat was weighed, and the fat mass of the inguinal groin of each group was calculated. Average

The data was presented in ±SD mode and statistically analyzed by one-way ANOVA. Statistical results are indicated by symbols or English letters. Different symbols or letters indicate statistical differences between groups (p < 0.05). The same symbols or letters indicate no statistical difference between groups (p > 0.05).

Please refer to FIG. 1A and FIG. 1B. FIG. 1A is a bar graph of the effect of subcutaneous injection of no-cell resveratrol subcutaneous injection and micro-free green tea extract on subcutaneous fat content in rats. Fig. 1B is a bar graph of the effect of subcutaneous injection of resveratrol without a microcell and the subcutaneous injection of a non-micronized green tea extract on the relative total weight gain of rats. Wherein, the amount of the lower inguinal fat is the sum of the fat mass of the inferior groin on the left and right sides.

The results in Figure 1A show that after three subcutaneous fat injections, the local fat mass at the injection site of the resveratrol group and the green tea extract group was not significantly reduced compared with the high-fat control group (p>0.05), showing direct Injection of the dissolved resveratrol or green tea extract into the subcutaneous fat layer does not reduce the local fat at the injection site. Figure IB shows that after three subcutaneous fat injections, the weight of the rats in the resveratrol group and the green tea extract group was not significantly reduced compared with the high-fat control group (p>0.05), indicating that it would directly The dissolved resveratrol or green tea extract is injected into the subcutaneous fat layer and does not reduce body weight.

Among them, green tea extract is an excellent water-soluble component. Previous cell experiments have found that it can promote the apoptosis of fat cells, but the green tea extract is dissolved and injected directly into the subcutaneous fat, which can not reduce its local subcutaneous fat. It is also impossible to lose weight. It can be seen from this experiment that direct injection of resveratrol or green tea extract into the subcutaneous fat layer is not sufficient to reduce local fat and body weight. In order to overcome this problem, the inventors have further studied and developed the pharmaceutical composition of the present invention comprising resveratrol.

Experiment 2: Effect of different kinds of resveratrol subcutaneous injection on subcutaneous fat mass and body weight of rats The resveratrol physiological saline solution, resveratrol PEG solution, and resveratrol ELP solution were prepared in the following manner.

Preparation method of resveratrol physiological saline solution:

500 mg of resveratrol was mixed with an appropriate amount of physiological saline for injection to make a final volume of 100 mL. Stirring was uniform, and resveratrol was completely dissolved to obtain a resveratrol physiological saline solution, and the concentration of resveratrol in the aqueous resveratrol physiological saline solution was 5 mg/mL.

Preparation method of resveratrol PEG solution:

15 g of polyethylene glycol 400 (PEG400), 15 g of glycerol, and an appropriate amount of physiological saline for injection were mixed to make a final volume of 100 mL. Stir well, completely dissolve the polyethylene glycol 400 and glycerin to obtain a polyethylene glycol and glycerin mixture. Mix 450 mg of resveratrol with a suitable amount of polyethylene glycol and glycerin to give a final volume of 90 ml. Stir well and completely dissolve resveratrol to obtain resveratrol PEG solution. The concentration of resveratrol in the resveratrol PEG solution was 5 mg/mL.

Preparation method of resveratrol ELP solution:

Mix 500 mg of resveratrol with 100 160 mL of dichloromethane and stir at room temperature at 150-500 rpm until resveratrol is completely dissolved. Add 20 g of polyoxyethylene 35 castor oil (Kolliphor ELP, simply called ELP) and stir evenly at 100 300 rpm to volatilize methylene chloride. After the dichloromethane is completely evaporated, the physiological saline solution for injection is slowly added to make the final volume up to 100 mL, and the mixture is uniformly stirred to obtain a resveratrol ELP solution. The concentration of resveratrol in the resveratrol ELP solution is 5 mg/mL, the concentration of polyoxyethylene 35 castor oil (ELP) is about 20% (% by weight;), and resveratrol and polyoxyethylene The weight ratio of 35 castor oil is 1:40.

Experiments were performed using a 6-week old SD strain male rat (male Sprague-Dawley rat). First, 20 rats were fed with high-fat diet (Research Diets, Inc.; model #D12492) to induce subcutaneous fat booster, and the rats were continuously fed to a body weight of 330±10 g. Rats were randomly divided into 4 groups: control group, physiological saline group, PEG group and ELP group, with 5 rats in each group, so that there was no statistical difference in body weight of each group. The body weight of each rat was recorded and defined as the "pre-test weight" of each rat. Then, the drug is administered in the following manner.

The resveratrol physiological saline solution, the resveratrol PEG solution, and the resveratrol ELP solution were respectively injected into the subcutaneous fat layer of the inguinal region of the physiological saline group, the PEG group, and the ELP group, each injection. Amount per metric 4 kg (4 mL/kg) was injected into the body weight, so that each dose was 20 mg resveratrol per kg body weight (20 mg/kg _{; the} calculation method was 4 mL/kg χ 5 mg/mL = 20 mg/ Kg), the control group was given the same volume of physiological saline for injection in the same injection method as above.

The above injection site was the lower inguinal fat of rats, which was injected on the left and right sides on average, and was injected once on the first, second, third and fourth days of the test. High-fat diets were continuously administered during the trial, and changes in body weight were recorded daily. Drinks were recorded once a week for a total of 14 days. Rats were sacrificed with carbon dioxide on day 15.

The body weight of each rat was recorded and defined as the "post-test weight" of each rat. The "post-test weight" was subtracted from the "post-test weight" of each rat to obtain "total weight gain". The total weight gain of each group of rats was divided by the total weight gain of the control group rats to obtain "relative total weight gain".

The subcutaneous fat in the lower groin of the left and right sides of the rat was weighed, and the subcutaneous fat in the lower and inferior groin was added to calculate the subcutaneous fat mass in the inferior groin. The subcutaneous fat mass of the inferior groin of each group of rats was divided by the subcutaneous fat mass of the inferior groin of the control group, and the "relative weight of subcutaneous fat in the lower inguinal region" was obtained.

Data were presented as mean ± 80 and statistically analyzed by one-way ANOVA. Statistical results are indicated by symbols or English letters. Different symbols or letters indicate statistical differences between groups (p < 0.05), and the same symbols or letters indicate no statistical difference between groups (p > 0.05).

Please refer to Figure 2A and Figure 2B. Figure 2A is a bar graph of the effect of subcutaneous injection of resveratrol prepared by different excipients on the amount of subcutaneous fat in rats. Fig. 2B is a bar graph of the effect of subcutaneous injection of resveratrol prepared by different excipients on the relative total weight gain of rats.

The results of Fig. 2A showed that the relative weight of the subcutaneous fat in the lower inguinal region of the control group was 100±27.6%, and the relative weight of the subcutaneous fat in the lower inguinal region of the saline group was 108.2±24.7%, and the lower inguinal area of the PEG group was subcutaneously. The relative weight of fat was 114.0±4.4%, and the relative weight of subcutaneous fat in the lower inguinal region of the ELP group was 72.5±0.0%. There was no significant difference between the relative weight of the inguinal subcutaneous fat of the rats in the saline group and the control group, indicating that the resveratrol was directly injected into the subcutaneous fat layer at the application site, and the fat at the application site could not be reduced (local fat; ). There was no significant difference in the relative weight of subcutaneous fat in the lower inguinal region between the PEG group and the control group. The relative weight of the subcutaneous fat in the lower inguinal region of the ELP group was significantly different from that in the control group (p<0.05). In the ELP group, the relative weight of the subcutaneous fat in the lower inguinal region was reduced by 27.5%.

The results in Figure 2B show that the relative total weight gain of the control group rats was 100.0 ± 30.8 %, the relative total weight gain of the rats in the saline group was 128.3 ± 16.9%, and the relative total weight gain of the rats in the PEG group was 120.8 ± 18.2%, the relative total weight gain of the ELP group was 101.3±22.0%, and there was no significant difference between the four groups (p>0.05).

As can be seen from the above experiment, direct injection of resveratrol into the subcutaneous fat layer at the application site does not reduce the fat (local fat) at the application site, nor does it reduce body weight. Injecting a resveratrol composition containing an excipient PEG (a common co-solvent;) into the subcutaneous fat layer at the application site does not reduce the fat at the application site (local fat;), nor does it reduce body weight, but will The resveratrol composition to which the nonionic surfactant ELP is added is injected to the subcutaneous fat layer at the application site, but the fat at the application site (local fat;) can be remarkably reduced. Therefore, it is necessary to further investigate whether the resveratrol composition must contain a nonionic surfactant in order to reduce subcutaneous fat (local fat) at the application site and reduce body weight. Further analysis revealed that the above-mentioned resveratrol PEG solution had no micelles, and the resveratrol ELP solution had micelles, and resveratrol was coated in the micelles formed by ELP. Therefore, it is more necessary to further explore the effects of micelles on reducing local fat and reducing body weight.

Experiment 3: Effect of subcutaneous injection of resveratrol single component containing microvesicles on subcutaneous fat mass and body weight in rats

The resveratrol ELP partial microcyst dosage form, the resveratrol HS-15 partial microcyst dosage form, the resveratrol ELP microcyst dosage form, and the resveratrol HS-15 microcyte dosage form were prepared in the following manner.

Preparation method of resveratrol ELP partial microcyst dosage form: Mix 20 g of polyoxyethylene 35 castor oil (ie, ELP) with an appropriate amount of physiological saline for injection, and make the final weight reach 100 ° evenly, so that polyoxyethylene 35 Castor oil (ie ELP) was completely dissolved and a 20% ELP solution was obtained. Mix 400 mg of resveratrol with an appropriate amount of 20% ELP solution to a final weight of 80 g. Stirring evenly, so that resveratrol is completely dissolved, the resveratrol ELP partial microcyst dosage form can be obtained. The concentration of resveratrol in the resveratrol ELP partial microcyst dosage form is about 5 mg/mL, the concentration of polyoxyethylene 35 castor oil (ELP) is about 20% (% by weight;), and resveratrol The weight ratio to polyoxyethylene 35 castor oil is about 1:40.

Method for preparing resveratrol HS-15 partial microcyst dosage form: mixing 20 g of polyethylene glycol stearate 15 (Kolliphor HS 15, abbreviated as HS-15) with an appropriate amount of physiological saline for injection to make the final Weighs up to 100 g. Stir well and completely dissolve the polyethylene glycol stearate 15 (BP HS-15;) to obtain a 20% HS-15 solution. Mix 400 mg of resveratrol with an appropriate amount of 20% HS-15 solution to a final weight of 80 g. Stir well and completely dissolve resveratrol to obtain resveratrol HS-15 partial microcyst dosage form. The concentration of resveratrol in the resveratrol HS-15 partial microcyst dosage form is about 5 mg/mL, and the concentration of polyethylene glycol stearate 15 (ie, HS-15) is about 20% (% by weight) And the weight ratio of resveratrol to polyethylene glycol stearate 15 (ie, HS-15) is about 1:40.

Method for preparing resveratrol ELP microcyst dosage form: The same procedure as for the resveratrol ELP solution in Experiment 2.

Resveratrol Preparation of HS-15 microcyst dosage form: Mix 500 mg of resveratrol with 80 140 mL of dichloromethane and stir at room temperature at 150-500 rpm until resveratrol is completely dissolved. Add 20 g of polyethylene glycol stearate 15 (Kolliphor HS 15, abbreviated as HS-15), stir evenly at a speed of 100 ~ 300 rpm to volatilize methylene chloride. After the dichloromethane is completely evaporated, the physiological saline solution for injection is slowly added to make the final volume up to 100 g, and the mixture is uniformly stirred to form a plurality of drug-containing micelles, thereby obtaining a resveratrol HS-15 microcyst form. The concentration of resveratrol in the resveratrol HS-15 microcyst dosage form is about 5 mg/g, and the concentration of polyethylene glycol stearate 15 (HS-15) is 20% (% by weight;), And the weight ratio of resveratrol to polyethylene glycol stearate 15 (HS-15) is 1:40.

Determination of resveratrol ELP partial microcyst dosage form, resveratrol using a particle size analyzer

The HS-15 partial microcyst dosage form, the resveratrol ELP microcyst dosage form, and the resveratrol HS-15 microcyte dosage form contain micelles and measure the particle size of the micelles.

The results showed that the resveratrol ELP partial microcyst form and the resveratrol HS-15 partial microcyte form contained a large amount of precipitate, and the number of drug-containing micelles was small. The resveratrol ELP microcyst dosage form and the resveratrol HS-15 microcyte dosage form are clear and non-stratified, and the number of drug-containing micelles is large. It can be seen that although the resveratrol ELP partial microcyst form and the resveratrol HS-15 partial microcyst form contain a large amount of precipitate, the supernatant still contains micelles, therefore, resveratrol The ELP partial microcyte dosage form, the resveratrol HS-15 partial microcyte dosage form, the resveratrol ELP microcyte dosage form, and the resveratrol HS-15 microcyte dosage form are all pharmaceutical compositions of the present invention.

Experiments were performed using a 6-week old SD strain male rat (male Sprague-Dawley rat). First, with high fat diet

(High-fat diet, the label is Research Diets, Inc.; model #D 12492) fed 20 rats to induce subcutaneous fat booster, and after continuous feeding until the rat weighed 330±10 g, the rats were randomized. Divided into 5 groups, which were control group, ELP partial microcell group, HS-15 partial microcell group, ELP microcell group, and HS-15 microcell group, 4 rats in each group, so that the weight of each group of rats There are no statistical differences. The body weight of each rat was recorded and defined as the "pre-test weight" of each rat. Then, the drug is administered in the following manner.

The resveratrol ELP partial microcyst dosage form, the resveratrol HS-15 partial microcyst dosage form, the resveratrol ELP microcyst dosage form, and the resveratrol HS-15 microcyst dosage form are separately mixed (make part) The precipitate in the microcytose formulation was uniformly suspended;), and injected into the subcutaneous fat layer of the inferior groin of the ELP partial microcell group, the HS-15 partial microcell group, the ELP microcell group, and the HS-15 microcell group, respectively. Each injection was injected with 4 mL (4 mL/kg) per kilogram of body weight, so that each dose was 20 mg of resveratrol C20 mg/kg per kilogram of body weight; the calculation method was 4 mL/kg χ 5 mg /mL = 20 mg/kg), the control group was given the same volume of physiological saline for injection in the same injection method as above.

The above injection site was the lower inguinal fat of rats, which was injected on the left and right sides on average, and was injected once on the first, second, third, fourth, fifth, and sixth days of the test. High-fat diets were continuously administered during the test, and changes in body weight were recorded daily. Drinks were recorded once a week for a total of 14 days. Rats were sacrificed with carbon dioxide on day 15.

According to the preparation method of the above dosage form and the particle size analysis result, the ELP concentration and the resveratrol concentration in the resveratrol ELP partial microcyst dosage form and the resveratrol ELP microcyst dosage form are the same, and only the microcapsules are contained. The quantity varies. Therefore, compared with the resveratrol ELP partial microcyte dosage form, if the resveratrol ELP microcyst dosage form significantly reduces the local fat at the application site, it means that the formation of drug-containing microvesicles is a resveratrol composition which can significantly reduce the application site. A key factor for topical fat; if the resveratrol ELP microcyte dosage form results in a significant reduction in body weight, it is representative that the formation of drug-containing micelles is a key factor in the significant reduction in body weight of the resveratrol composition.

Similarly, the HS-15 concentration of the resveratrol HS-15 partial microcyte dosage form and the resveratrol HS-15 microcyte dosage form, The resveratrol concentrations were consistent and the number of drug-containing micelles was different. Therefore, compared with the resveratrol HS-15 partial microcyte dosage form, if the resveratrol HS-15 microcyst dosage form causes a significant decrease in local fat at the application site, it means that the formation of drug-containing microvesicles is a resveratrol composition. Significantly reduce the key factor of local fat in the application site; if the resveratrol HS-15 microcyte dosage form significantly reduces body weight, it means that the formation of drug-containing microvesicles is a key factor in the significant reduction of body weight of resveratrol composition.

The experimental results show that injection of the resveratrol ELP partial microcyst dosage form or the resveratrol HS-15 partial microcyst dosage form to the subcutaneous fat layer of the application site can reduce the fat (local fat) at the application site. On the other hand, injection of the resveratrol ELP microcyst form or the resveratrol HS-15 microcyst form into the subcutaneous fat layer of the application site can also reduce the fat (local fat) at the application site. Among the four dosage forms, the partial lipolysis effect of the resveratrol ELP microcyst dosage form was the best.

The resveratrol ELP microcyte formulation significantly reduced localized fat compared to the resveratrol ELP partial microcyte formulation. The resveratrol HS-15 microcyte formulation significantly reduced localized fat compared to the resveratrol HS-15 partial microcyte formulation.

It can be seen that the formation of the microcapsule is a key factor for the resveratrol composition to significantly reduce the local fat at the application site, and the partial lipid solution of the drug-containing micelle formed by the polyoxyethylene castor oil derivative (for example, ELP). best effect.

The results of the experiment showed that the injection of the resveratrol ELP partial microcyst dosage form or the resveratrol HS-15 partial microcyst dosage form to the subcutaneous fat layer at the application site reduced body weight. On the other hand, injection of the resveratrol ELP microcyst dosage form or the resveratrol HS-15 microcyst form into the subcutaneous fat layer of the application site can also reduce body weight. Among the four dosage forms, the resveratrol ELP microcyst dosage form has the best weight loss effect.

It is known from the experiment that the formation of the micelle is a key factor for the significant reduction of body weight of the resveratrol composition, and the drug-containing micelles containing the polyoxyethylene castor oil derivative (e.g., ELP) have the best weight loss effect.

Experiment 4: Preparation of a pharmaceutical composition for reducing local fat

In this experiment, a first pharmaceutical composition was prepared using resveratrol, and a second pharmaceutical composition was prepared using resveratrol and green tea extract.

The steps for preparing the first pharmaceutical composition are as follows:

(a) mixing the first weight of resveratrol with a solvent, stirring at 200 to 500 rpm at room temperature until the resveratrol is completely dissolved;

(b) adding a second weight of a pharmaceutically acceptable surfactant, stirring at a rotational speed of 100 300 rpm to volatilize the solvent, wherein the hydrophilic-lipophilic balance of the surfactant Value, HLB value) is greater than 10;

(c) after the solvent is completely volatilized, slowly add a third weight of the pharmaceutically acceptable aqueous solution, stir well, and obtain a plurality of drug-containing micelles;

(d) After filtering through a 0.2 μιη filter, the filtrate containing the drug-containing cells is stored in the dark;

Wherein, in the step (c), the drug-containing micelle is a micro-structure formed by a surfactant, and resveratrol is coated in the drug-containing micelle.

Preferably, the third weight is greater than or equal to 0g.

Preferably, in step (a), the boiling point of the solvent is less than the boiling point of pure water. Preferably, in the step (a), the solvent is a hydrophilic solvent.

Preferably, the hydrophilic solvent is at least one of methanol, ethanol, acetone, and other hydrophilic solvents, or a combination thereof. Preferably, the solvent in step (a) is a lipophilic solvent.

Preferably, the lipophilic solvent is at least one of ether, benzene, chloroform, ethyl acetate, chloroform, hexamethylene and other lipophilic vehicles or a combination thereof.

Preferably, in the step (b), the surfactant is a nonionic surfactant.

Preferably, the nonionic surfactant is polysorbate 80 (Tween 80), 2-hydroxyethyl 12-hydroxyoctadecanoate (solutol HS 15), polyoxyethylene castor oil derivatives ^ and At least one or a combination of other nonionic surfactants.

Preferably, the polyoxyethylene castor oil derivative is polyoxyethylene 35 castor oil (Crem ₀ ph ₀ r ELP), polyoxyethylene

At least one or a combination of 40 hydrogenated castor oil (Cremophor RH 40), and other polyoxyethylene castor oil derivatives.

Preferably, in steps (a) and (b), the weight ratio of the first weight of resveratrol to the second weight of surfactant is from 1:4 to 1:500.

Preferably, in steps (a) and (c), the weight ratio of the first weight of resveratrol to the third weight of the pharmaceutically acceptable aqueous solution is from 1:400 to 3:50.

Preferably, in the step (c), the pharmaceutically acceptable aqueous solution is water for injection, aqueous solution for injection, or physiological saline.

Preferably, in step (c), the pharmaceutically acceptable aqueous solution comprises a local anesthetic.

Preferably, the local anesthetic is at least one of an amide, a p-aminobenzamide, and an amino ether or a combination thereof.

Preferably, the amide is dibucaine; Lidocaine; mepivacaine hydrochloride (Mepivacaine HC1), bupivacine hydrochloride (Bupivacine HC1), pyrrole At least one or a combination of pyrrocaine HC1, prilocaine HCl, Digammacaine, and Oxethazaine.

Preferably, the p-aminobenzamide is at least one or a combination of butacaine; dimethocaine; and totocaine.

Preferably, the amino ether is at least one of quinicaine (Quinisocaine;) and pramocaine or a combination thereof.

Preferably, in step (c), the pharmaceutically acceptable aqueous solution contains an antioxidant.

Preferably, the antioxidant is beta-carotene; lutein; lycopene; bilirubin; vitamin K; Vitamin C (also known as ascorbic acid), vitamin E, uric acid, nitric oxide, nitroxide, pyruvate , catalase, superoxide dismutase, glutathione peroxidases, N-acetyl cysteine; and pomelo At least one or a combination of naringenin. The steps for preparing the second pharmaceutical composition are as follows:

(al) mixing a fourth weight of resveratrol with a solvent, stirring at 200 to 500 rpm at room temperature until the resveratrol is completely dissolved;

(bl) adding a fifth weight of a pharmaceutically acceptable surfactant, stirring uniformly at a rotational speed of 100 to 300 rpm to volatilize the solvent, wherein the hydrophilic-lipophilic balance of the surfactant (hydrophilic-lipophilic) Balance value, HLB value) is greater than 10;

(cl) after the solvent is completely volatilized, slowly add a sixth weight of the first pharmaceutically acceptable aqueous solution, and stir evenly at a rotational speed of 100 300 rpm to form a plurality of drug-containing micelles;

(dl) After filtering through a 0.2 μηη filter, the filtrate containing the drug-containing microcapsules is stored in the dark;

Wherein the first pharmaceutically acceptable aqueous solution comprises a seventh weight of green tea extract; the green tea extract comprises a first green tea extract component, and the first green tea extract component is epigallocatechin gallate.

Preferably, in step (al), the boiling point of the solvent is less than the boiling point of pure water.

Preferably, in the step (al), the solvent is a hydrophilic solvent.

Preferably, the hydrophilic solvent is at least one of methanol, ethanol, acetone, and other hydrophilic solvents, or a combination thereof. Preferably, the solvent in step (al) is a lipophilic solvent.

Preferably, in step (b1), the surfactant is a nonionic surfactant.

Preferably, the polyoxyethylene castor oil derivative is polyoxyethylene 35 castor oil (Crem ₀ ph ₀ r ELP), polyoxyethylene 40 hydrogenated castor oil (Cremophor RH 40), and other polyoxyethylene castor oil derivatives At least one of or a combination thereof.

Preferably, between step (cl) and step (dl), the method further comprises the steps of:

(cl l) An eighth weight of a second pharmaceutically acceptable aqueous solution is added and stirred uniformly to completely dissolve the second pharmaceutically acceptable aqueous solution.

Preferably, the green tea extract is dissolved in the first pharmaceutically acceptable aqueous solution, the medicated microcell is a micro-structure formed by a surfactant, and resveratrol is coated in the In the drug-containing micelles.

Preferably, the content of epigallocatechin gallate in the green tea extract in the first pharmaceutically acceptable aqueous solution is from 100 to 100 based on 100% by weight of the total weight of the green tea extract. %.

Preferably, in steps (al) and (cl), the weight ratio of the fourth weight of resveratrol to the seventh weight of the green tea extract is from 30:1 to 1:30.

Preferably, in steps (al) to (cl), the total weight of the fourth weight of resveratrol and the seventh weight of the green tea extract is one weight unit, and the fifth weight is surface active. The weight of the agent is 0.24 70 units by weight; or alternatively, the weight ratio of the total weight of the resveratrol to the green tea extract to the surfactant is from 4:1 to 1:70. Preferably, in steps (al), (cl), and (cl l), the total weight of the fourth weight of resveratrol and the seventh weight of the green tea extract is one unit of weight, The total weight of the sixth weight of the first pharmaceutically acceptable aqueous solution and the eighth weight of the second pharmaceutically acceptable aqueous solution is 16,400 weight units; or is the sum of the fourth weight and the seventh weight : The sum of the sixth weight and the eighth weight is 1:400 3:50.

The total weight of the resveratrol and the green tea extract is one unit of weight, and the total weight of the first pharmaceutically acceptable aqueous solution and the second pharmaceutically acceptable aqueous solution is 10 1000 weight units; Yes, the total weight of the resveratrol and the green tea extract: the total weight of the first pharmaceutically acceptable aqueous solution and the second pharmaceutically acceptable aqueous solution is from 1:1000 to 1:10.

Preferably, in the steps (cl) and (cl l), the first pharmaceutically acceptable aqueous solution and the second pharmaceutically acceptable aqueous solution are water for injection, aqueous solution for injection, or physiological saline.

Preferably, in step (cl), the first pharmaceutically acceptable aqueous solution comprises a local anesthetic.

Preferably, in step (cl), the pharmaceutically acceptable aqueous solution contains an antioxidant.

Preferably, the antioxidant is beta-carotene, clutein, lycopene, bilirubin, vitamin K, vitamin C. Also known as ascorbic acid, vitamin E, uric acid, nitric oxide, nitroxide, pyruvate, catalase (catalase), superoxide dismutase, glutathione peroxidases, N-acetyl cysteine; and naringenin At least one of or a combination thereof.

In this experiment, a third pharmaceutical composition is prepared using resveratrol and a water-soluble drug, and a fourth pharmaceutical composition and a fifth pharmaceutical composition are prepared using resveratrol and other fat-soluble drugs.

The steps for preparing the third pharmaceutical composition are as follows:

(a2) mixing resveratrol with a solvent, stirring at 200-500 rpm at room temperature until the resveratrol is completely dissolved; (b2) adding a pharmaceutically acceptable surfactant at a speed of 100-300 rpm Stirring uniformly, the solvent is volatilized, wherein the surfactant has a hydrophilic-lipophilic balance value, HLB value;) is greater than 10; (c2) after the solvent is completely volatilized, slowly adding the first pharmaceutically acceptable aqueous solution, stirring uniformly at a rotational speed of 100 300 rpm to form a plurality of drug-containing micelles;

(d2) After filtering through a 0.2 μιη filter, the filtrate containing the drug-containing microcapsules is stored in the dark;

Wherein the first pharmaceutically acceptable aqueous solution comprises a water soluble drug.

Preferably, the first pharmaceutically acceptable aqueous solution comprises a local anesthetic.

Preferably, the pharmaceutically acceptable aqueous solution contains an antioxidant.

Preferably, the antioxidant is beta-carotene, lutein, lycopene, bilirubin, vitamin K, vitamin C (vitamin K), vitamin C (vitamin K), vitamin C (vitamin K), vitamin C (vitamin K), vitamin C (vitamin K) Vitamin C; also known as ascorbic acid, vitamin E, uric acid, nitric oxide, nitroxide, pyruvate, peroxidation Catalase, superoxide dismutase, glutathione peroxidases, N-acetyl cysteine; and naringenin At least one of naringenin) or a combination thereof.

Preferably, in the step (a2), the boiling point of the solvent is smaller than the boiling point of the pure water.

Preferably, in the step (a2), the solvent is a hydrophilic solvent.

Preferably, the hydrophilic solvent is at least one of methanol, ethanol, acetone, and other hydrophilic solvents, or a combination thereof. Preferably, the solvent in step (a2) is a lipophilic solvent.

Preferably, in the step (b2), the surfactant is a nonionic surfactant.

Preferably, the nonionic surfactant is polysorbate 80 (Tween 80), 2-hydroxyethyl

At least one or a combination of 12-hydroxyoctadecanoate (solutol HS 15), polyoxyethylene castor oil derivatives, and other nonionic surfactants.

Preferably, between step (c2) and step (d2), the method further comprises the steps of: (c21) A second pharmaceutically acceptable aqueous solution is added and stirred well to completely dissolve the second pharmaceutically acceptable aqueous solution.

Preferably, the water-soluble drug is dissolved in the first pharmaceutically acceptable aqueous solution, the drug-containing microcell is a micro-structure formed by a surfactant, and resveratrol is coated in the In the drug cell.

Preferably, the water-soluble drug in the first pharmaceutically acceptable aqueous solution is green tea extract, epigallocatechin gallate, epicchinchin, epicatechin gall. Epicatechin gallate, Epigallocatechin, Gallocatechin gallate, Gallocatechin, Catechin gallate, catechin (Catechin;), epigallocatechin gallate (EGCG)^ caffeine (Caffeine;), carnitine (Carnitine; also known as carnitine or carnitine), L-carnitine (L- At least one or a combination of carnitine;), Synephrine^ Chi orogenic acid, and other water soluble drugs.

Preferably, in steps (a2) and (c2), the weight ratio of the resveratrol to the water-soluble drug is from 30:1 to 1:30. Preferably, in steps (a2) to (c2), the total weight of the resveratrol and the water-soluble drug is one unit of weight, and the weight of the surfactant is 0.24 70 weight units; The weight ratio of the total weight of the resveratrol to the water-soluble drug to the surfactant is from 4:1 to 1:70.

Preferably, in the steps (a2), (c2), and (c21), the first pharmaceutically acceptable aqueous solution is based on the total weight of the resveratrol and the water-soluble drug in one weight unit. The total weight of the second pharmaceutically acceptable aqueous solution is 16-400 weight units; or is the sum of the fourth weight and the seventh weight: the sum of the sixth weight and the eighth weight is 1:400 ~3:50.

Preferably, in the steps (c2) and (c21), the first pharmaceutically acceptable aqueous solution and the second pharmaceutically acceptable aqueous solution are water for injection, aqueous solution for injection, or physiological saline.

The steps for preparing the fourth pharmaceutical composition are as follows:

(A) a step of preparing a drug-containing micro cell composition for preparing a drug-containing micro cell composition;

(B) a step of preparing a second liposoluble drug microcapsule composition for preparing a second liposoluble drug cell sub-composition:

(C) mixing the drug-containing microcapsule composition with the second lipophilic drug microcapsule composition to prepare the fourth pharmaceutical composition;

Wherein the step (A) of preparing the drug-containing micro-cell composition comprises the following steps (a3) to (d3) _:

(a3) mixing resveratrol with the first solvent, stirring at 200 to 500 rpm at room temperature until the resveratrol is completely dissolved; (b3) adding a pharmaceutically acceptable first surfactant at a rotation speed of 100 - 300 Stirring under rpm conditions, the first solvent is volatilized, wherein the first surfactant has a hydrophilic-lipophilic balance value (HLB value) greater than 10;

(c3) after the first solvent is completely volatilized, slowly adding a pharmaceutically acceptable aqueous solution, and stirring uniformly to form a plurality of drug-containing micelles;

(d3) after filtering through a 0.2 μηη filter, the filtrate is the drug-containing microcapsule composition containing the drug-containing micelle; Moreover, the step (B) of preparing the second fat-soluble drug microcapsule composition comprises the following steps (a4) to (d4):

(a4) mixing a further fat-soluble drug with the second solvent, and stirring at 200 to 500 rpm at room temperature until the other fat-soluble drug is completely dissolved;

(b4) adding a pharmaceutically acceptable second surfactant, stirring uniformly at a rotation speed of 100 - 300 rpm to volatilize the second solvent, wherein the second surfactant has a hydrophilic-lipophilic balance value (hydrophilic- Lipophilic balance value, HLB value) is greater than 10;

(c4) after the second solvent is completely volatilized, slowly adding a pharmaceutically acceptable aqueous solution, and stirring uniformly to form a plurality of second fat-soluble drug micelles;

(d4) After filtering through a 0.2 μηη filter, the filtrate is the second liposoluble drug cell sub-composition containing the second liposoluble drug micelle.

Wherein, in the step (c3), the drug-containing micelle is a micro-structure formed by the first surfactant, and resveratrol is coated in the drug-containing micelle. In the step (c4), the second fat-soluble drug micelle is a micro-structure formed by the second surfactant, and the other fat-soluble drug is coated in the second fat-soluble drug cell.

Preferably, in step (a3) or / and step (a4), the boiling point of the first solvent or / and the second solvent is less than the boiling point of pure water. Preferably, in the step (a3) or / and the step (a4), the first solvent or the second solvent is a hydrophilic solvent.

Preferably, the hydrophilic solvent is at least one of methanol, ethanol, acetone, and other hydrophilic solvents, or a combination thereof. Preferably, the first solvent or / and the second solvent in the step (a3) or / and (a4) are lipophilic solvents.

Preferably, in the step (b3) or / and (b4), the first surfactant or / and the second surfactant are nonionic surfactants.

Preferably, in steps (a3) and (b3), the weight ratio of the resveratrol to the first surfactant is from 1:4 to 1:500.

Preferably, in steps (a4) and (b4), the weight ratio of the other fat-soluble drug to the second surfactant is from 1:4 to 1:500.

Preferably, in steps (a3) and (c3), the weight ratio of the resveratrol to the pharmaceutically acceptable aqueous solution is from 1:400 to 3:50.

Preferably, in steps (a4) and (c4), the weight ratio of the other fat-soluble drug to the pharmaceutically acceptable aqueous solution is 1: 400 to 3: 50.

Preferably, in the step (c3) or / and (c4), the pharmaceutically acceptable aqueous solution is water for injection, aqueous solution for injection, or physiological saline.

Preferably, in step (c3) or / and (c4), the pharmaceutically acceptable aqueous solution comprises a local anesthetic.

Preferably, in the step (c3) or / and (c4), the pharmaceutically acceptable aqueous solution contains an antioxidant.

The steps for preparing the fifth pharmaceutical composition are as follows:

(a5) mixing resveratrol and other fat-soluble drugs with a solvent, and stirring at 200 to 500 rpm at room temperature until completely dissolved;

(b5) adding a pharmaceutically acceptable surfactant, stirring at a rotation speed of 100 - 300 rpm to volatilize the solvent, wherein the surfactant has a hydrophilic-lipophilic balance value, HLB value ;) is greater than 10;

(c5) after the solvent is completely volatilized, slowly adding a pharmaceutically acceptable aqueous solution, and stirring uniformly to form a plurality of drug-containing micelles and a plurality of second fat-soluble drug micelles;

(d5) After filtering through a 0.2 μm filter, the filtrate containing the drug-containing micelles and the plurality of second fat-soluble drug micelles was stored in a dark place.

The range of the solvent, the surfactant, the pharmaceutically acceptable aqueous solution, and other fat-soluble drugs used in the fifth pharmaceutical composition is the same as that of the fourth pharmaceutical composition. Further, the range of the proportional relationship between the components in the fifth pharmaceutical composition is also the same as that of the fourth pharmaceutical composition.

Preferably, the pharmaceutically acceptable aqueous solution comprises a local anesthetic or/and an antioxidant. Preferably, the range of the type of the local anesthetic or/and the antioxidant used in the fifth pharmaceutical composition is the same as that of the fourth pharmaceutical composition.

Experiment 5: Determination of the quality of pharmaceutical compositions

Experiment 5-1 Component Analysis

The pharmaceutical composition is allowed to stand for at least 20 minutes, and if delamination does not occur, it is further tested by a particle size analyzer. The micelle was contained in the pharmaceutical composition by a particle size analyzer. If the pharmaceutical composition is analyzed by a particle size analyzer, the measured particle diameter is less than 250 nm, and the PDI value is less than 0.4. The solution in the pharmaceutical composition is visually observed to be clear and transparent, and the optical path can be observed after the laser irradiation solution is used. , on behalf of the pharmaceutical composition has a microcell.

If the pharmaceutical composition has a microcell, the composition prepared is the medical composition of the present invention which can be used for reducing local fat.

If the pharmaceutical composition is not layered after standing and has micelles, the prepared composition is a preferred pharmaceutical composition of the present invention which can be used for reducing topical fat.

Experiment 5-2 Analysis of stability of pharmaceutical compositions using particle size distribution

The particle size distribution (purchasing from Malvern) was used to determine the distribution of the particle size and the polydispersity index (PDI). If the polydispersity index is less than 0.4, the stability of the pharmaceutical composition is good. That is, the micelles in the pharmaceutical composition can exist stably.

Experiment 5-3 The stability of the pharmaceutical composition was determined by an accelerated stability test.

The pharmaceutical composition of the present invention has a storage condition of 2 to 8 °C. In order to test the stability of the pharmaceutical composition, the inventors placed the pharmaceutical composition in a relatively high temperature and high humidity environment (temperature 25 ° C ± 2 ° C, relative humidity RH 60% ± 5%), observed in the pharmaceutical composition How long can the microvesicles be stable in a relatively high temperature state to predict how long the pharmaceutical composition can be stored at 2-8 °C.

If the pharmaceutical composition can be stored for n months at 25 ° C, the pharmaceutical composition can be stored at 5 ° C for a period of time of 2 ⁽⁽²⁵ - ^{5) / 1Q)} times n months. That is, the pharmaceutical composition can be stored at a temperature of 5 ° C for a length of ²² times, that is, 4 times.

For example, if the pharmaceutical composition can be stored for 6 months at 25 ° C, the pharmaceutical composition can be stored for a period of 24 months at 5 ° C (6 months X 4 times = 24 month).

Preferably, the pharmaceutical composition is maintained in a precipitate-free manner when subjected to an accelerated test under the conditions of a temperature of 25 ± 2 degrees (25 ± 2 ° C;), a relative humidity of RH 60% ± 5%, and avoiding direct light. The state is at least 24 hours.

Preferably, the pharmaceutical composition is maintained in a state free of precipitates for at least 6 months when the accelerated test is carried out under the conditions of a temperature of 25 ± 2 degrees Celsius, a relative humidity of RH 60% ± 5%, and direct light exposure.

Preferably, the pharmaceutical composition is maintained in a state free of precipitates for at least 24 months at a temperature of 2 to 8 °C.

Experiment 6: Maximum drug loading of drug-containing micelles formed by various nonionic surfactants

Since the maximum drug loading of the drug-containing cells directly affects the injection volume, the local subcutaneous fat layer (such as the subcutaneous surface of the face) The fat layer) has a huge impact on the volume, side effects, and burden of the drug that must be contained in a single dose. Therefore, this experiment will investigate the maximum drug loading of drug-containing micelles formed by various nonionic surfactants to evaluate which nonionic surfactant is the best excipient for the preparation of the pharmaceutical composition of the present invention. .

Four nonionic surfactants were used for this experiment. The four nonionic surfactants are polyoxyethylene 35 castor oil (ie ELP), polyethylene glycol stearate 15 (ie HS-15), polyoxyethylene 40 hydrogenated castor oil (Cremophor RH 40, Referred to as RH 40), and polysorbate 80 (Tween 80).

The experiment was divided into 4 groups, namely ELP group, HS-15 group, RH40 group, and Tween 80 group.

Experimental steps:

(a') Mix 2.0 g (example of the first weight) resveratrol with 300 500 mL of dichloromethane, and stir at 150-500 rpm at room temperature until the resveratrol is completely dissolved;

(b') adding 18.0 g (example of the second weight) of a single of the above nonionic surfactants, stirring at a rotation speed of 100 - 300 rpm to volatilize methylene chloride;

(C) After the solvent is completely volatilized, a composition is obtained, 20 g in total; 2 g of the composition is taken, and 8 g is slowly added (example of the third weight;) physiological saline for injection is stirred uniformly to obtain a composition to be tested. Things. The concentration of resveratrol in the composition to be tested was 20 mg/g, and the concentration of the nonionic surfactant was 18%.

The components to be tested in the ELP group, the HS-15 group, the RH40 group, and the Tween 80 group were allowed to stand for at least 20 minutes to observe whether stratification occurred. If delamination occurs, the concentration of resveratrol is too high to cause the microcells in the first stage composition to rupture, that is, the concentration of resveratrol cannot be prepared by using the nonionic surfactant. g of the pharmaceutical composition of the invention.

Assessing the toxicity of various excipients (ELP, HS-15, RH40, and Tween 80), according to national pharmacopoeias

(Pharmacopoeia) The excipient concentration limit for excipients is calculated, and the drug loading limit of the drug-containing micelles formed by various excipients is estimated.

In order to know the maximum drug loading limit of ELP, the inventors conducted the following experiment 7. It was found that the maximum drug loading of ELP was greater than or equal to 166.7 mg/g (when the ratio of resveratrol to ELP was 1:5, prepared) The pharmaceutical composition contains 166.7 mg/g resveratrol;).

Experimental results show that ELP is the preferred excipient for the preparation of the pharmaceutical compositions of the present invention. The concentration of resveratrol in the pharmaceutical composition prepared by ELP can reach 166.7 mg/g.

Experiment 7: Preparation of pharmaceutical compositions using polyoxyethylene 35 castor oil (ELP)

In this experiment, a series of pharmaceutical compositions of the present invention were prepared by using the ratio change of resveratrol and polyoxyethylene 35 castor oil (ELP), and stability analysis was carried out to know resveratrol and polyoxyethylene 35. The appropriate ratio of castor oil (ELP) and the maximum drug loading when the pharmaceutical composition of the present invention is prepared by ELP.

The experiment was divided into 9 groups, namely groups 1 to 9. The preparation method of the pharmaceutical composition of each group was substantially the same as the experimental procedure of experiment 6, only the weight of resveratrol (the first weight in step (a'); The weight of the ELP (the second weight in the step (b');), the weight of the physiological saline for injection (the third weight in the step (c');) is different. In this experiment, the principle of adding the weight of resveratrol (first weight;), the weight of ELP (second weight;), and the weight of physiological saline for injection (third weight) are shown in Table 1. In this experiment, the ratio of the resveratrol to ELP in the 1st to 9th groups (weight ratio) is one to one (1:1), one to two five (1: 2.5), one to five (1: 5), one to eight (1:8), one to ten (1:10), one to forty (1:40), one to eighty (1:80), one to two hundred (1:200) And one to five hundred (1:500), and the final concentration of resveratrol in the pharmaceutical composition prepared in groups 1 to 9 is 1000 mg/g, 285.71 mg/g, and 166. 7 mg/ g, 60 mg/g, 30 mg/g, 7.5 mg/g, 3.75 mg/g, 0.5 mg/g, 0.2 mg/g. That is, in the preparation method of the pharmaceutical composition of the first to the ninth groups, the weight ratio of the resveratrol in the step ( _a ') to the ELP in the step ( _b ') (the ratio of the first weight to the second weight; ) in order of one to one, one to two five, one to five, one to eight, one to ten, one to forty, one to eighty, one to two hundred, and one to five hundred, and in steps (c') After adding a third weight of physiological saline for injection, the final concentrations of resveratrol are sequentially prepared at 1000 mg/g, 285.71 mg/g, 166.7 mg/g, 60 mg/g, 30 mg/ g, 7.5 mg/g, 3.75 mg/g, 0.5 mg/g, and 0.2 mg/g of pharmaceutical composition. Wherein, when the final concentration of the drug is expressed in mg/g, it represents the number of milligrams of resveratrol contained per gram of the pharmaceutical composition.

A particle size analyzer was used to determine whether or not a micelle was contained in the pharmaceutical composition, and the particle size of the micelle was measured.

The particle size distribution and the polydispersity index (PDI) were measured using a particle size analyzer to evaluate the stability of the pharmaceutical composition. The resveratrol content in the micelles was analyzed by high performance liquid chromatography (HPLC; for example, HPLC-UV) and defined as "initial drug content".

The accelerated stability test was used to observe whether the medicinal composition was stratified under high temperature storage conditions (25±2 degrees Celsius; 3 months), and high performance liquid chromatography (HPLC); for example, HPLC -UV) Analyze the drug content in the micelles, defined as "the drug content after the accelerated experiment". Divide the "drug content after accelerated experiment" by "starting drug content" to get "percentage of drug content". If the percentage of the drug content is greater than or equal to 95%, it represents an excellent stability of the pharmaceutical composition. Table 1 Sample preparation table for preparing pharmaceutical composition using ELP

White peony in the composition of resveratrol and ELP

(weight ratio;) final concentration of rosin (mg/g)

1 1 : 1 1000

2 1 :2.5 285.71

3 1 :5 166.7

4 1 : 8 60 1:40

1:80 3.75

1:200

1:500 0.2

Please refer to Table 2, Table 2 for the stability analysis results of pharmaceutical compositions. Table 2 shows that when the weight ratio of resveratrol to ELP is 1:5 to 1:500, there are microcells in each group of pharmaceutical compositions, and the measured cell size is 10 250 nm, therefore, chalk The pharmaceutical composition prepared by the ratio of rusol to ELP of 1:5 to 1:500 is a pharmaceutical composition which can be used for reducing local fat in the present invention.

In terms of stability, when the ratio of resveratrol to ELP is 1:5 to 1:500, the PDI is less than 0.4. Therefore, in order to prepare a pharmaceutical composition which is preferably used for reducing the local fat, the weight of the ELP should be greater than or equal to 5 weight units, based on the weight of the resveratrol. Preferably, the weight of the resveratrol is 1 weight unit, and the weight of the ELP is 5 500 weight units. Preferably, the weight of the resveratrol is one unit of weight, and the weight of the ELP is 10 80 units by weight. Preferably, the weight of the resveratrol is 1 unit by weight, and the weight of the ELP is 8 to 80 units by weight. Table 2 Stability analysis of pharmaceutical compositions

After accelerating the experiment, resveratrol and ELP cells were accelerated after the experiment.

PDI drug content ratio (weight ratio) particle size (nm) appearance

Percentage C1⁄4)

1:1 404.13 ± 64.70 1.00 ± 0.00

1:2. 787.07 ± 186.84 0.71 ± 0.50

240.63 ± 9.49 0.189 ± 0.06 Clarification without delamination 97.41 ±0.17

30 1 : 8 174 ± 1.97 0.15 ± 0.05

1 : 10 15.61 ± 0.39 0.180 ± 0.01 Clarified without delamination 98.32 ± 0.23

1 : 40 13.66 ± 0.49 0.237 ± 0.03 Clear blue without delamination 95.76 ± 0.66

1 : 80 12.22 ± 0.13 0.181 ± 0.0 Clarification without delamination 95.54 ± 0.03

1 : 200 12.84 ± 0.29 0.17 ± 0.02

1 : 500 12.08 ± 0.96 0.15 ± 0.09 In the above table, the blank field indicates that no analysis has been performed. According to the data in Table 2, the third and fifth to seventh groups of pharmaceutical compositions were stored in an environment of 25 degrees Celsius for 3 months, and the percentage of resveratrol drug content in each group of samples was greater than 95%, and the initial There was no significant downward trend in drug content. From the results, it is understood that the pharmaceutical composition has good stability, and it is estimated according to the empirical rule of the accelerated experiment that the pharmaceutical compositions can be stored for at least 24 months under refrigeration at 2 to 8 °C.

Experiment 8: Effect of Preparation of Pharmaceutical Compositions on Polyoxyethylene 35 Castor Oil on Local Liquefaction and Weight Reduction

Example 1 : Preparation of resveratrol pharmaceutical composition using polyoxyethylene 35 castor oil and resveratrol

(a') Mix 0.4 g (example of the first weight) resveratrol with 20 70 mL of ethanol, and stir at 200-500 rpm at room temperature until the resveratrol is completely dissolved;

(b') Add 4 g (the second weight of the example) polyoxyethylene 35 castor oil, stir evenly at 100-300 rpm, and evaporate the ethanol;

(C) After the ethanol is completely evaporated, the physiological saline for injection is slowly added so that the weight of the physiological saline for injection reaches 80 g (example of the third weight;), and the mixture is uniformly stirred to form a plurality of drug-containing micelles;

(d') After filtering through a 0.2 μηη filter, the filtrate is a pharmaceutical composition of resveratrol containing drug-containing micelles. Store in the dark.

In this embodiment, the specific gravity of the physiological saline for injection is lg/mL. Therefore, the volume of the physiological saline solution for injection of 80 g is 80 mL. Accordingly, the concentration of resveratrol in the pharmaceutical composition of resveratrol is about 5 mg/mL (0.4 g ÷ 80 mL = 0.005 g/mL). = 5 mg/mL) ₀

In this embodiment, the weight ratio of the first weight of resveratrol to the second weight of polyoxyethylene 35 castor oil (surfactant) is 0.4:4, i.e., the weight ratio is 1:10.

In this embodiment, the weight ratio of the first weight of resveratrol to the third weight of the physiological saline for injection is 0.4: 80, that is, the weight ratio is 1: 200ο

Example 2: Preparation of resveratrol compound medicine composition using polyoxyethylene 35 castor oil, resveratrol and green tea extract

(al') Mix 0.36 g (fourth weight example) resveratrol with 20-70 mL ethanol, and stir at room temperature at 200-500 rpm until resveratrol is completely dissolved;

(bl') Add 4 g (5th weight example) polyoxyethylene 35 castor oil, stir evenly at 100-300 rpm, and evaporate the ethanol;

(cl') after the ethanol is completely volatilized, slowly adding a sixth weight of the first injection physiological saline, and uniformly stirring at a rotation speed of 100 to 300 rpm to form a plurality of drug-containing micelles, wherein the first injection 0.04 g (the seventh-weight example) green tea extract is contained in physiological saline, and the composition of the green tea extract contains epigallocatechin gallate;

(cir) adding a second weight of the second injection physiological saline, so that the total volume of the physiological saline for injection is so mL (that is, the sixth weight of the first injection physiological saline and the eighth weight second The total volume of physiological saline for injection was 80 mL), and the mixture was evenly stirred to completely dissolve the physiological saline solution for the second injection.

(dl') After filtering through a 0.2 μηη filter, the filtrate containing the drug-containing microvesicles is a resveratrol compound pharmaceutical composition. Store in cold storage.

In this embodiment, the concentration of resveratrol and green tea extract in the resveratrol combination medicinal composition is about 5 mg/mL [(0.36 g + 0.04 g) ÷ 80 mL = 0.4 g ÷ 80 mL = 0.005 g/mL = 5 mg/mL].

In this embodiment, if the total weight of the green tea extract (0.04 g) is 100% by weight, the content of epigallocatechin gallate in the green tea extract is 95%.

In this embodiment, the weight ratio of the fourth weight of resveratrol to the seventh weight of the green tea extract is 0.36: 0.04, i.e., the weight ratio is 9:1.

In this embodiment, the fourth weight of the fourth weight of resveratrol and the seventh weight of the green tea extract (0.36 g + 0.04 g = 0.4 g) is one weight unit. The weight of the surfactant is 10 weight units (4g ÷ 0.4g = 10).

In this embodiment, the specific gravity of the physiological saline for injection is 1 g/mL. Therefore, in the specific embodiment, the total weight of the sixth weight of the first injection physiological saline and the eighth weight second physiological saline is 80 mL, and the total weight of the physiological saline is 80 g. .

According to this, the total weight of the fourth weight of resveratrol and the seventh weight of the green tea extract (0.36g + 0.04g = o.4g) is one unit of weight, the first of the sixth weight The total weight (80 g) of the physiological saline for injection and the eighth weight of the physiological saline for injection was 200 weight units (80 g ÷ 0.4 g = 200).

Example 3: Lipolysis effect of pharmaceutical composition of resveratrol

Experiments were performed using a 6-week old SD strain male rat (male Sprague-Dawley rat). First, 15 rats fed with high-fat diet induced an increase in subcutaneous fat. After continuous feeding to a rat weighing 330±10 g, the rats were randomly divided into 3 groups: high-fat control group and resveratrol low-dose. Group, and high-dose resveratrol group, 5 rats per group, There was no statistical difference in body weight between the groups.

The 5 mg/mL resveratrol pharmaceutical composition of the above Example 1 was injected into the subcutaneous fat layer of the inguinal region of the rats in the low dose group of resveratrol, and the injection amount was 10 mg per kg body weight (10 mg per dose). /kg) Resveratrol; that is, 2 mL of the 5 mg/mL resveratrol pharmaceutical composition of the above Example 1 was injected per kg of body weight. The 5 mg/mL resveratrol pharmaceutical composition of the above Example 1 was injected into the subcutaneous fat layer of the inguinal region of the rats in the high dose group of resveratrol, and the injection amount was 20 mg per kg body weight (20 mg per injection). /kg) Resveratrol; that is, 4 mL of the 5 mg/mL resveratrol pharmaceutical composition of the above Example 1 was injected per kg of body weight. In the high-fat control group, physiological saline solution for the same volume injection was administered by the same injection method as described above.

The above injection site was the lower inguinal fat of rats, which was injected on the left and right sides on average, and was injected once on the first, second, third and fourth days of the test. During the test period, high-fat diet was continuously administered, and changes in body weight were recorded daily. Drinking water was recorded once a week, fasted on the 14th day of the test, and rats were sacrificed with carbon dioxide on the 15th day.

The fat in the lower groin of the left and right sides of the rat was weighed, and the fat mass of the inguinal groin of each group was calculated. Data were presented as mean ± SD and statistically analyzed by one-way ANOVA. Statistical results are indicated by symbols or English letters. Different symbols or letters indicate statistical differences between groups (p < 0.05). The same symbols or letters indicate no statistical difference between groups (p > 0.05).

Please refer to Figure 3 and Table 3. Figure 3 is a bar graph of the effect of the pharmaceutical composition of resveratrol on the amount of subcutaneous fat in rats. Table 3 shows the extent to which the pharmaceutical composition of resveratrol reduces the amount of subcutaneous fat in rats. Among them, the subcutaneous fat mass in the inferior groin is the sum of the fat weights on the left and right sides.

The experimental results showed that compared with the high-fat control group, the lower inguinal fat mass of the rats in the low-dose resveratrol group (administered at 10 mg/kg) had a decreasing trend, and the percentage of local fat reduction was 5.6%. In the high-dose resveratrol group (administered at 20 mg/kg), the subcutaneous fat mass in the lower inguinal region was significantly lower than that in the high-fat control group (p<0.05), and the local fat was reduced. The percentage reached 18.1%. Table 3. The degree of reduction of subcutaneous fat in rats by the pharmaceutical composition of resveratrol

Subcutaneous fat in the lower inguinal (g) percentage of fat reduction

Mean Shi SD (%) high fat control group. 4± 0.8 resveratrol low dose group. 1± 0.4.6% resveratrol high dose group 4.4± 0.8 18.1%

According to the experience of the inventors, when the frequency of administration to rats is 4 times, the frequency of administration for humans is 1 to 12 Times. Preferably, the frequency of administration to humans is from 1 to 12 times per interval from 1 day to 30 days. Preferably, the frequency of administration to humans is from 1 to 6 times per interval from 1 day to 30 days.

According to the inventor's experience, when administered to rats at doses ranging from 10 mg/kg to 20 mg/kg, the dosage for humans is 0.2 40 mg/kg.

Preferably, the dosage administered to humans is 0.4 20 mg/kg.

Preferably, the dose applied to humans is 0.2 to 20 mg per square centimeter.

Preferably, the dose applied to humans is 0.4 to 12 mg per square centimeter.

Example 4: Lipolysis and weight loss effects of resveratrol compound pharmaceutical composition

Preparation of Green Tea Extract Composition: The green tea extract was dissolved using physiological saline for injection to prepare a 5 mg/mL green tea extract composition.

Experiments were performed using a 6-week old SD strain male rat (male Sprague-Dawley rat). First, 20 rats were fed with high-fat diet to induce an increase in subcutaneous fat. After continuous feeding to a rat weighing 330±10 g, the rats were randomly divided into 4 groups: high-fat control group, green tea extract group, and white. The resveratrol group and the resveratrol-green tea extract compound group, 5 rats in each group, showed no statistical difference in body weight of each group of rats. The body weight of each rat was recorded and defined as the "pre-test weight" of each rat. Then, the drug is administered in the following manner.

The 5 mg/mL green tea extract composition of the present example was injected into the subcutaneous fat layer of the inguinal region of the green tea extract group, and each injection was injected with 10 mg (10 mg/kg) of green tea extract per kg of body weight. That is, 2 mL of the 5 mg/mL green tea extract composition of this example was injected per kg of body weight. The 5 mg/mL resveratrol pharmaceutical composition of the above Example 1 was injected into the subcutaneous fat layer of the inguinal region of the rats in the resveratrol group, and the injection amount was 10 mg per kg body weight (10 mg/kg). Resveratrol; that is, 2 mL of the 5 mg/mL resveratrol pharmaceutical composition of the above Example 1 was injected per kg of body weight. The 5 mg/mL resveratrol combination medicinal composition of the above Example 2 was injected into the subcutaneous fat layer of the inguinal region of the resveratrol-green tea extract compound group, and the injection amount was 10 per kg of body weight per injection. Mg (10 mg/kg) resveratrol and green tea; that is, 2 mL of the 5 mg/mL resveratrol combination medicinal composition of the above Example 2 was injected per kg of body weight. In the high-fat control group, the same volume of physiological saline for injection was administered in the same manner as described above.

Since each group was given 10 mg/kg of the drug, the resveratrol-green tea extract compound group was partially localized. The lipolysis effect should be between the resveratrol group and the green tea extract group. If the resveratrol-green tea extract compound group has better partial lipolysis effect than the resveratrol and green tea extract group, it represents resveratrol and green tea extract in resveratrol-complex medicine composition. having a synergistic effect _(sy nerg y) on the efficacy of local fat soluble.

Please refer to Figure 4A and Table 4. Figure 4A is a bar graph of the effect of resveratrol combination medicinal composition on subcutaneous fat mass in rats. Table 4 shows the degree of reduction of subcutaneous fat in rats by the compound composition of resveratrol. Among them, the subcutaneous fat mass of the inferior groin is the sum of the fat weights of the left and right sides.

The results of the experiment in Fig. 4A showed that the subcutaneous fat at the injection site of the green tea extract group was not reduced as compared with the high fat control group (the administration dose was 10 mg of green tea extract per kg body weight). There was no statistical difference in the amount of subcutaneous fat at the injection site of the rats in the resveratrol group compared with the high-fat control group, but the percentage of local fat reduction was 5.6% (the dose was 10 mg per kg body weight). Resveratrol). The subcutaneous fat content at the injection site of the resveratrol-green tea extract group showed a significant decrease (p<0.05), and the local fat reduction percentage reached 18.9% (the dose was 10 mg white per kg body weight). Resveratrol and green tea extract;). That is, the resveratrol compound medicine composition can significantly achieve the effect of reducing local fat, which is 3.4 times that of the resveratrol pharmaceutical composition.

This example shows a combination pharmaceutical composition of resveratrol plus green tea extract of the present invention which has a better partial lipolysis effect than the same dose of resveratrol.

Comparing the partial lipolysis effect of the resveratrol group, the green tea extract group, and the resveratrol-green tea extract compound group, the resveratrol and green tea extract in the resveratrol compound medicine composition are known. There is a synergy in the efficacy of topical lipolysis. Table 4, Resveratrol compound pharmaceutical composition reduces the amount of subcutaneous fat in rats

Subcutaneous fat in the lower inguinal (g) percentage of fat reduction

Mean Shi SD (%) high fat control group 5.4 ± 0.8 - green tea extract group 5.4 ± 0.9 0% resveratrol group 5.1 ± 0.4 5.6% resveratrol - green tea

4.4 ± 0.6 18.9% Extract Compound Group See Figure 4B and Table 5. Figure 4B is a bar graph of the effect of resveratrol combination medicinal composition on relative total weight gain in rats. Table 5 shows the degree of weight loss in rats treated with resveratrol compound medicinal composition.

The experimental results in Figure 4B show that the relative total weight gain of the green tea extract group was not reduced compared with the high fat control group. Less (administered dose of 10 mg of green tea extract per kg of body weight;). The relative total weight gain of the resveratrol group was statistically different from that of the high-fat control group, but it showed a decreasing trend. The percentage of local fat reduction was 7.4% (the dose was 10 mg of chalk per kg body weight). Resin;). Compared with the high-fat control group, the percentage of relative total weight gain in the resveratrol-green tea extract group was 15.9% (administration dose was 10 mg resveratrol and green tea extract per kg body weight); ), although there is still no significant difference, the effect is better than the resveratrol group. According to the experience of the inventors, as long as the dosage or frequency of administration is increased, a significant weight loss effect can be achieved by using the resveratrol single-medical composition prepared by the present invention and the resveratrol-green tea extract compound medicine group. Table 5. Resveratrol compound pharmaceutical composition to reduce the degree of weight loss in rats

Percentage of weight loss Group Relative total weight gain (%)

(%) High-fat control group 100.0± 16.3 - Green tea extract group 103.7± 21.8 0% Resveratrol group 92.6± 17.8 7.4% Resveratrol-green tea extract

84.1± 0.6 15.9%

Compound Compound Group Experiment 9: Effect of compound pharmaceutical composition formed by resveratrol and other fat-soluble drugs on lipolysis

In this experiment, resveratrol and other fat-soluble drugs were used to prepare a compound pharmaceutical composition to evaluate the fat-dissolving effect of various fat-soluble compound medicine compositions on mature fat cells.

In this experiment, _puera rin and quercetin were used to prepare various fat-soluble compound pharmaceutical compositions. Experiment 9-1 Cytotoxicity test

Cell viability assay (MTT assay) was used to assess whether 50 ppm of resveratrol, puerarin, or quercetin is toxic to cells other than adipocytes, and if not toxic, test.

The results showed that 50 ppm of resveratrol, puerarin, and quercetin were not cytotoxic to normal somatic cells other than rat adipocytes, so this dose would not be normal for the body. Cells cause effects.

Experiment 9-2 Fat-dissolving effect of mature fat cells

The DMSO control group cell culture solution, resveratrol cell culture solution, puerarin cell culture solution, quercetin cell culture solution, resveratrol-puerarin compound cell culture solution, and resveratrol were prepared in the following manner. Quercetin compound cell culture fluid. DMSO control group Cell culture medium: DMSO was mixed with an appropriate amount of sterile water to prepare a 0.5% DMSO solution. A 0.5% DMSO solution was mixed with a cell culture solution (product name: Dulbecco's Modified Eagle Medium, available from Gibco) to prepare a DMSO control group cell culture solution, wherein the volume ratio of the 0.5% DMSO solution to the cell culture solution was 1 : 1000.

Resveratrol cell culture solution: Resveratrol is mixed with an appropriate amount of 0.5% DMSO solution to prepare a solution of resveratrol. The resveratrol solution was mixed with a cell culture solution (product name: Dulbecco's Modified Eagle Medium, available from Gibco) to prepare a resveratrol cell culture solution containing 50 ppm of resveratrol, wherein resveratrol The volume ratio of the solution to the cell culture medium was 1:1000.

Puerarin cell culture solution: Puerarin (purchased from Sigma-Aldrich) was mixed with an appropriate amount of 0.5% DMSO solution to prepare a puerarin solution. The puerarin solution was mixed with the cell culture solution to prepare a puerarin cell culture solution containing 50 ppm of puerarin, wherein the volume ratio of the puerarin solution to the cell culture solution was 1:1000.

Quercetin cell culture solution: Quercetin (purchased from Sigma-Aldrich) was mixed with an appropriate amount of 0.5% DMSO solution to prepare a quercetin solution. The quercetin solution was mixed with the cell culture solution to prepare a quercetin cell culture solution containing 50 ppm of quercetin, wherein the volume ratio of the puerarin solution to the cell culture solution was 1:1000.

Resveratrol-puerarin compound cell culture solution: Resveratrol, puerarin, and an appropriate amount of 0.5% DMSO solution are mixed to prepare a resveratrol-puerarin compound solution. Among them, the weight ratio of resveratrol to puerarin is 2:3. The resveratrol-puerarin compound solution is mixed with the cell culture solution to prepare a cell culture solution containing 50 ppm of resveratrol-puerarin compound drug, wherein the concentration of resveratrol is 20 ppm, puerarin The concentration was 30 ppm, and the volume ratio of the resveratrol-puerarin complex solution to the cell culture solution was 1:1000.

Resveratrol-quercetin compound cell culture solution: Resveratrol, quercetin, and an appropriate amount of 0.5% DMSO solution are mixed to prepare a resveratrol-quercetin compound solution. Among them, the weight ratio of resveratrol to quercetin is 2:3. The resveratrol-quercetin compound solution is mixed with the cell culture solution to prepare a cell culture solution containing 50 ppm of resveratrol-quercetin compound drug, wherein the concentration of resveratrol is 20 ppm. The concentration of quercetin was 30 ppm, and the volume ratio of the resveratrol-quercetin compound solution to the cell culture solution was 1:1000.

Experimental steps for fat-dissolving effect of mature fat cells:

3T3-L1 precursor fat cells (purchased from Taiwan Food Industry Development Research Institute, abbreviated as BCRC) were inoculated into 12 well plates to contain 1 X 10 ⁵ cells per well. After 2 days of culture, a cell-differentiated culture medium (DMI medium) containing 0.5 μΜ ΙΒΜΧ (purchased from Sigma-Aldrich), 0.1 μΜ Dexamethasone (purchased from Sigma-Aldrich), and 5 μMιη Insulin (purchased from Humunlin R) was used. .)) Culture for 2 days. Then, it was cultured for 6 days with a medium containing 5 μm of insulin (Insulin), and when the cell type changed from a spindle shape to a spherical shape and many lipo droplets accumulated in the cells, it was differentiated into mature fat cells. (Mature adipocytes;).

The mature adipocytes were divided into 6 groups: DMSO control group, resveratrol group, puerarin group, quercetin group, resveratrol-puerarin compound group, and resveratrol-quercetin compound group. .

DMSO control group cell culture medium, resveratrol cell culture solution, puerarin cell culture solution, quercetin cell culture solution, resveratrol-puerarin compound cell culture solution, and resveratrol-quercetin Compound cell culture solution, Mature fat cells in the DMSO control group, the resveratrol group, the puerarin group, the quercetin group, the resveratrol-puerarin combination group, and the resveratrol-quercetin combination group were cultured for 24 hours.

After mixing Annexin V protein (purchased from eBioscience) and Propidium iodide dye (abbreviated as PI; purchased from eBioscience) with each group of cells for a period of time, flow cytometry was used to analyze each group of cells by Annexin V. The ratio of protein and PI staining label to assess the proportion of mature adipocytes for apoptosis. Among them, mature adipocytes, which are simultaneously labeled with Annexin V protein and PI stain, represent that they have entered the apoptotic program; the more mature adipocytes undergo apoptosis, the better the lipolysis effect of the administered drugs is, and represents Lipolysis is through apoptotic procedures rather than necrosis;

Since the total dose of each group of drugs administered was 50 ppm, the apoptosis effect of the resveratrol-puerarin combination group should be between the resveratrol group and the puerarin group. If the apoptosis effect of resveratrol- puerarin compound group is better than that of resveratrol group and puerarin group, it represents resveratrol and puerarin in resveratrol-puerarin compound medicine composition. The effect of lipids has a synergistic effect ( _Syner gy). Similarly, the apoptosis effect of the resveratrol-quercetin combination group should be between the resveratrol group and the quercetin group. If the resveratrol-quercetin combination group has better apoptosis than the resveratrol group and the quercetin group, it represents resveratrol and bismuth in the resveratrol-quercetin compound pharmaceutical composition. Peelin has a synergistic effect on the efficacy of lipolysis.

Please refer to Figure 5. Figure 5 shows the effect of resveratrol-other fat-soluble drug combination on apoptosis of mature adipocytes.

The results in Figure 5 show that the percentage of apoptosis in the DMSO control group was 4.9 ± 2.5%, the percentage of apoptosis in the resveratrol group was 19.0 ± 1.1%, and the percentage of apoptosis in the puerarin group was 7.2 ± 3.7%. The percentage of apoptosis was 5.9±2.6%, the percentage of apoptosis in the resveratrol-puerarin group was 50.6±3.8%, and the percentage of apoptosis in the resveratrol-quercetin combination group was 12.1±2.7%.

Comparing the apoptosis effects of the resveratrol group, the puerarin group, and the resveratrol-puerarin compound group, the resveratrol-puerarin compound medicine composition has a synergistic effect on the efficacy of the lipolysis (sy _nerg y).

It can be seen that the compound pharmaceutical composition formed by resveratrol and various fat-soluble drugs can achieve the fat-dissolving effect, and the synergistic effect of resveratrol and various fat-soluble drugs on the fat-soluble effect (sy _nerg) y). Therefore, the present invention utilizes resveratrol and various fat-soluble drugs to prepare drug-containing micelles and second fat-soluble drug micelles, thereby preparing resveratrol-other fat-soluble drug compound medicine composition, which can be used as a local A pharmaceutical composition that dissolves fat and reduces weight.

Experiment 10: Effect of compound pharmaceutical composition formed by resveratrol and water-soluble drugs on lipolysis

In this experiment, a water-soluble drug other than green tea extract and resveratrol were used to prepare a compound pharmaceutical composition to evaluate the fat-dissolving effect of various resveratrol-water-soluble drug compound pharmaceutical compositions on mature fat cells.

In this experiment, caffeine (caffeine;) and L-camitine were used to prepare various resveratrol-water-soluble drug compound pharmaceutical compositions. Experiment 10-1 Cytotoxicity test

The cell viability assay (MTT assay) was used to assess whether 50 ppm caffeine (caffeine;), and L-camitine were toxic to cells other than adipocytes, and if not toxic, the lipolysis test was performed.

The results showed that 50 ppm of caffeine (caffeine;) and L-carnitine were not cytotoxic to normal somatic cells other than rat adipocytes, so this dose did not affect normal somatic cells.

Experiment 10-2 Fat-dissolving effect of mature fat cells

Preparing sterile water control group cell culture medium, resveratrol cell culture solution, caffeine cell culture solution, L-carnitine cell culture solution, resveratrol-caffeine compound cell culture solution, and white gourd in the following manner Alcohol-L-carnitine compound cell culture solution.

Aseptic water control group Cell culture medium: Sterile water is mixed with the cell culture medium to prepare a sterile water control group cell culture solution. Wherein, the volume ratio of the sterile water to the cell culture solution is 1:1000.

Resveratrol cell culture solution: The same procedure as for the resveratrol cell culture solution in Experiment 9-2.

Caffeine Cell Culture Solution: Caffeine (purchased from Sigma-Aldrich) was mixed with an appropriate amount of sterile water to prepare a caffeine solution. The caffeine solution was mixed with the cell culture solution to prepare a caffeine cell culture solution containing 50 ppm of caffeine, wherein the volume ratio of the caffeine solution to the cell culture solution was 1:1000.

L-carnitine cell culture solution: L-carnitine (purchased from Sigma-Aldrich) was mixed with an appropriate amount of sterile water to prepare a L-carnitine solution. The L-carnitine solution was mixed with the cell culture solution to prepare a L-carnitine cell culture solution containing 50 ppm of L-carnitine, wherein the volume ratio of the L-carnitine solution to the cell culture solution was 1:1000.

Resveratrol-caffeine compound cell culture solution: Resveratrol, caffeine, and an appropriate amount of sterile water are mixed to prepare a resveratrol-caffeine compound solution. Among them, the weight ratio of resveratrol to caffeine is 2:3. The resveratrol-caffeine compound solution is mixed with the cell culture solution to prepare a cell culture solution containing 50 ppm of resveratrol-caffeine compound, wherein the concentration of resveratrol is 20 ppm, caffeine The concentration was 30 ppm, and the volume ratio of resveratrol-caffeine complex solution to cell culture solution was 1:1000.

Resveratrol-L-carnitine compound cell culture solution: Resveratrol, L-carnitine, and an appropriate amount of sterile water are mixed to prepare a resveratrol-L-carnitine compound solution. Wherein, the weight ratio of resveratrol to L-carnitine is 2:3. The resveratrol-L-carnitine compound solution is mixed with the cell culture solution to prepare a cell culture solution containing 50 ppm of resveratrol-L-carnitine compound drug, wherein the concentration of resveratrol is 20 ppm. The concentration of L-carnitine was 30 ppm, and the volume ratio of the resveratrol-L-carnitine compound solution to the cell culture solution was 1:1000.

The method for preparing mature adipocytes was the same as in Experiment 9-2.

The mature adipocytes were divided into 6 groups: sterile water control group, resveratrol group, caffeine group, L-carnitine group, resveratrol-caffeine compound group, and resveratrol-L-carnitine. Compound group.

Aseptic water control group cell culture solution, resveratrol cell culture solution, caffeine cell culture solution, L-carnitine cell culture solution, resveratrol-caffeine compound cell culture solution, and resveratrol-left-handed Carnitine compound cell culture medium, respectively, cultured sterile water control group, resveratrol group, caffeine group, L-carnitine group, resveratrol-caffeine compound group, and resveratrol-L-carnitine compound Mature fat cells in the group for 24 hours. After mixing Annexin V protein (purchased from eBioscience) and Propidium iodide dye (referred to as PI; purchased from eBioscience) with each group of cells for a period of time, flow cytometry was used to analyze the cells of each group by Annexin V protein and PI. The ratio of the label of the dye to evaluate the proportion of mature adipocytes for apoptosis. Among them, mature adipocytes, which are simultaneously labeled with Annexin V protein and PI stain, represent that they have entered the apoptotic program; the more mature adipocytes undergo apoptosis, the better the lipolysis effect of the administered drugs is, and represents Lipolysis is through apoptotic procedures rather than necrosis;

Since the total dose of each group of drugs is 50 ppm, and the proportion of resveratrol is 40% and caffeine is 60%, the apoptosis effect of the resveratrol-caffeine compound group should be close. The average value of the resveratrol group and the caffeine group. If the apoptosis effect of the resveratrol-caffeine compound group is significantly better than that of the resveratrol group and the caffeine group, it represents resveratrol in the resveratrol-caffeine compound pharmaceutical composition. And caffeine has a synergistic effect on the efficacy of lipolysis. Similarly, since the total dose of each group of drugs is 50 ppm, and the proportion of resveratrol is 40% and L-carnitine is 60%, the cells of the resveratrol-L-carnitine compound group The effect of death should be close to the average of the resveratrol group and the L-carnitine group. If the resveratrol-L-carnitine compound group has a significantly better apoptosis effect than the resveratrol group and the L-carnitine group, it represents the white peony in the resveratrol-L-carnitine compound medicine composition. Resveratrol and L-carnitine have a synergistic effect on the efficacy of _lipolysis ( _Syner gy).

See Figure 6. Figure 6 shows the effect of resveratrol-other water-soluble drug combination medicinal composition on the decline of mature adipocytes.

The results in Figure 6 show that the percentage of apoptosis in the sterile water control group is 9.6 ± 1.5%, the percentage of apoptosis in the resveratrol group is 19.0 ± 1.1%, and the percentage of apoptosis in the caffeine group is 6.9 ± 1.1%. The percentage of apoptosis in the alkali group was 5.2±1.2%, the percentage of apoptosis in the resveratrol-caffeine group was 43.1±4.5%, and the percentage of apoptosis in the resveratrol-L-carnitine group was 19.3±0.5%. .

Comparing the apoptosis effects of the resveratrol group, the caffeine group, and the resveratrol-caffeine compound group, resveratrol and caffeine in the resveratrol-caffeine compound medicine composition are dissolved. Synergistic effect on the efficacy of lipids

(synergy)

Comparing the apoptosis effects of the resveratrol group, the L-carnitine group, and the resveratrol-L-carnitine compound group, resveratrol and levorotin in the resveratrol-L-carnitine compound medicine composition are known. Carnitine has a synergistic effect on the efficacy of lipolysis.

It can be seen that the compound pharmaceutical composition formed by resveratrol and various water-soluble drugs can achieve the fat-dissolving effect, and the resveratrol and various water-soluble drugs have a synergistic effect on the fat-soluble effect (sy _nerg) y). Therefore, the present invention utilizes resveratrol and various water-soluble drugs to prepare a resveratrol-water-soluble pharmaceutical compound pharmaceutical composition comprising a drug-containing microcell, which can be used as a pharmaceutical composition for partial fatliquoring and weight loss.

According to an embodiment of the present invention, the first pharmaceutical composition, the second pharmaceutical composition, the third pharmaceutical composition, the fourth pharmaceutical composition, the fifth pharmaceutical composition, and other pharmaceutical compositions provided by the present invention are provided by the present invention. , can reduce the amount of local fat. Therefore, the first pharmaceutical composition, the second pharmaceutical composition, the third pharmaceutical composition, the fourth pharmaceutical composition, the fifth pharmaceutical composition, and other pharmaceutical compositions provided by the present invention, which are provided by the present invention, can be used for the production. Preparation of subcutaneous implants, subcutaneous implants, implantable infusions, ointment or salve, or patches, which can be implanted subcutaneously, implanted, applied or applied. The percutaneous absorption method is applied to a site where it is desired to reduce subcutaneous fat.

Preferably, the first pharmaceutical composition, the second pharmaceutical composition, the third pharmaceutical composition, the fourth pharmaceutical composition, the fifth pharmaceutical composition, and other pharmaceutical compositions provided by the present invention are provided by the present invention. Subcutaneous fat injection method reduces the fat at the application site. Therefore, the first pharmaceutical composition, the second pharmaceutical composition, the third pharmaceutical composition, the fourth pharmaceutical composition, the fifth pharmaceutical composition, and other pharmaceutical compositions provided by the present invention, which are provided by the present invention, can be used for preparation An injection or subcutaneous injection for subcutaneous fat layer to reduce local subcutaneous fat.

The above are only the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Therefore, various other modifications or retouchings, etc., which are not departing from the spirit of the present invention, should be included in Within the claims of the present invention.

Claims

Rights request

1. A pharmaceutical composition for reducing localized fat, comprising:

a plurality of drug-containing micelles;

Resveratrol coated in the drug-containing micelle;

Wherein the drug-containing micelle is a micro-structure formed by a pharmaceutically acceptable polyoxyethylene castor oil derivative, and the polyoxyethylene castor oil derivative is hydrophilic and lipophilic The hydrophilic-lipophilic balance value (HLB value) is greater than 10.

The pharmaceutical composition according to claim 1, wherein the drug-containing micelles have a particle diameter of 3 to 250 nm.

The pharmaceutical composition according to claim 1, wherein the polyoxyethylene castor oil derivative is polyoxyethylene 35 castor oil (Cremophor ELP) ^ polyoxyethylene 40 hydrogenated castor oil (Cremophor RH 40), and other polyoxygen At least one or a combination of ethylene castor oil derivatives.

4. The pharmaceutical composition according to claim 1, wherein the weight ratio of the resveratrol to the surfactant is 1:4 to 1:500.

The pharmaceutical composition according to claim 4, wherein the weight ratio of the resveratrol to the surfactant is from 1:5 to 1:80.

The pharmaceutical composition according to claim 1, wherein the concentration of the resveratrol in the pharmaceutical composition is 0.2 to 166.7 mg/mL.

The pharmaceutical composition according to claim 6, wherein the concentration of the resveratrol in the pharmaceutical composition is 2.5 to 60 mg/mL.

The pharmaceutical composition according to claim 1, which further comprises a pharmaceutically acceptable aqueous solution.

The pharmaceutical composition according to claim 1 or 8, further comprising a second fat-soluble drug microcell; the second liposoluble drug microcell is a second polyoxyethylene castor oil derivative Another micro-structure is formed, and a second lipophilic drug is coated in the second liposoluble drug micelle.

The pharmaceutical composition according to claim 9, wherein the second polyoxyethylene castor oil derivative is polyoxyethylene 35 castor oil (Cremophor ELP;), polyoxyethylene 40 hydrogenated castor oil (Cremophor RH 40), and At least one or a combination of other polyoxyethylene castor oil derivatives.

The pharmaceutical composition according to claim 9, wherein the second fat-soluble drug is q _Uerce tin, sy _ne phri _ne , p _Uera rin, curcumin, And at least one or a combination of other fat-soluble drugs other than resveratrol.

12. The pharmaceutical composition according to claim 11, wherein the weight ratio of the resveratrol to the second fat-soluble drug is 30: 1 ~ 1: 20 ο

The pharmaceutical composition according to claim 1 or 8, which further comprises a water-soluble drug.

The pharmaceutical composition according to claim 13, wherein the water-soluble drug is green tea extract, epigallocatechin gallate, epicchinchin, epicatechin gallate. (Epicatechin gallate), Epigallocatechin, Gallocatechin gallate, Gallocatechin, Catechin gallate, Catechin; ), epigallocatechin gallate (EGCG)^ caffeine (Caffeine;), carnitine (Carnitine; also known as carnitine or carnitine), L-carnitine; At least one or a combination of sinverine (Synephrine;), chlorogenic acid ( Chi orogenic acid), and other water soluble drugs.

15. The pharmaceutical composition according to claim 14, wherein the weight ratio of the resveratrol to the water-soluble drug is 20:

1 ~ 1: 30.

The pharmaceutical composition according to claim 14, wherein the water-soluble drug is epigallocatechin gallate, and the concentration of epigallocatechin gallate in the pharmaceutical composition is 0.25 to 300 mg. /mL.

The pharmaceutical composition according to claim 14, wherein the water-soluble drug is a green tea extract, and the weight ratio of the resveratrol to the green tea extract is from 30:1 to 1:30.

18. A method of reducing the amount of subcutaneous fat in a localized portion of an individual comprising administering a pharmaceutical composition to the localized portion of the individual, wherein the pharmaceutical composition comprises:

a plurality of drug-containing micelles;

Resveratrol coated in the drug-containing micelle;

Wherein the drug-containing microcapsule is a micro-structure formed by a pharmaceutically acceptable nonionic surfactant, and the hydrophilic-lipophilic balance value of the nonionic surfactant is Chydrophilic-lipophilic balance value, HLB value) is greater than 10.

19. The method of claim 18, wherein the drug-containing micelles have a particle size of 3 250 nm.

The method according to claim 18, wherein the nonionic surfactant is polysorbate 80 (T _ween 80), 2-hydroxyethyl 12-hydroxyoctadecanoate (solutol HS 15), polyoxyethylene castor oil derivative

At least one of or combinations of polyoxyethylene castor oil derivatives; and other nonionic surfactants.

21. The method according to claim 20, wherein the polyoxyethylene castor oil derivative is polyoxyethylene 35 castor oil (Cremophor ELP) ^ polyoxyethylene 40 hydrogenated castor oil (Cremophor RH 40), and other polyoxyethylene oxime At least one or a combination of sesame oil derivatives.

22. The method according to claim 18, wherein the weight ratio of the resveratrol to the nonionic surfactant is from 1:4 to 1:500.

23. The method according to claim 18, wherein the concentration of the resveratrol in the pharmaceutical composition is 0.2 to 166.7 mg/mL.

24. The method of claim 18, further comprising a pharmaceutically acceptable aqueous solution.

The method according to claim 18 or 24, further comprising a second fat-soluble drug molecule cell; wherein the second fat-soluble drug molecule cell is formed by the second nonionic surfactant Another micro-structure, and a second fat-soluble drug is coated in the second fat-soluble drug micelle.

26. The method of claim 25, wherein the second nonionic surfactant has a hydrophilic-lipophilic balance value (HLB value) greater than 10.

27. The method of claim 26, wherein the second nonionic surfactant is polysorbate 80 (T _ween 80), 2-hydroxyethyl 12-hydroxyoctadecanoate (solutol HS 15), polyoxyethylene castor oil derived Object

28. The method of claim 27, the second nonionic surfactant is polyoxyethylene 35 castor oil (Cremophor ELP) ^ polyoxyethylene 40 hydrogenated castor oil (Cremophor RH 40), and other polyoxyethylene At least one of castor oil derivatives or a combination thereof.

29. The method according to claim 25, wherein the second fat-soluble drug is q _Uerce tin, synephrine, puerarin, curcuminoid and other whites. At least one or a combination of fat-soluble drugs other than resveratrol.

The method according to claim 29, wherein the weight ratio of the resveratrol to the second fat-soluble drug is from 30:1 to 1:20.

31. The method of claim 18 or 24, further comprising a water soluble drug.

32. The method according to claim 31, wherein the water-soluble drug is green tea extract, epigallocatechin gallate, epicchinchin, epicatechin gallate, epicatechin gallate (Epicatechin gallate), Epigallocatechin, Gallocatechin gallate, Gallocatechin, Catechin gallate, Catechin Catechin ^epigallocatechin gallate (EGCG;), caffeine (Caffeine;), carnitine (also known as carnitine or carnitine), L-carnitine (L) At least one or a combination of -camitine;), Synephrine; Chi orogenic acid, and other water soluble drugs.

33. The method of claim 32, wherein the weight ratio of resveratrol to the water soluble drug is from 20:1 to 1:30.

The method according to claim 32, wherein the water-soluble drug is epigallocatechin gallate, Further, the concentration of epigallocatechin gallate in the pharmaceutical composition is 0.25 to 300 mg/mL.

35. The method of claim 32, wherein the water soluble drug is a green tea extract and the weight ratio of the resveratrol to the green tea extract is from 30:1 to 1:30.

36. The method of claim 35, wherein the total weight of the resveratrol and the green tea extract is one weight unit, and the weight of the nonionic surfactant is 0.24 70 weight units; The weight ratio of the total weight of the resveratrol to the green tea extract to the nonionic surfactant is from 4:1 to 1:70.

37. The method of claim 18, wherein the pharmaceutical composition is administered at a localized dose of 0.2 to 20 mg per square centimeter.

38. The method of claim 18, wherein the pharmaceutical composition is administered at a localized dose of from 0.2 to 40 mg per kilogram.

39. The method of claim 18, wherein the pharmaceutical composition is administered at a frequency of from 1 to 12 administrations per interval from 1 day to 30 days.

40. The method of claim 18, wherein the individual is an animal or a human.

41. The method of claim 18, wherein the medical composition is injected, or applied to the localized portion of the individual.

42. The method of claim 18, further comprising a cosolvent, a suspending agent, and at least one oil phase excipients; Kind or a combination thereof.

43. The method of claim 42, wherein the oil phase excipient and at least one of the co-solvent together with the nonionic surfactant form the microstructure.